I.P. is funded by a Natural Sciences and Engineering Research Council of Canada grant (NSERC #418233-2012); a Fonds de Recherche du Qu&#233;bec-Sant&#233; (FRQS), a Quebec Breast Cancer Foundation career award, and a Leader Founds grant from the Canadian Foundation for Innovation grant. E.D. received a scholarship from the Fondation universitaire Armand-Frappier.

All animal protocols used in this study were approved by the University Animal Care Committee (INRS-Institut Armand-Frappier, Laval, Canada).
1. Identifying Protein Co-localization
<ol>
	<li>From tissue to microscopic slides 
	NOTE: Tissues and sections should be handled on dry ice.
	<ol>
		<li>Excise the mammary glands from an animal (for a complete description of this procedure, refer to Plante et al.)14.</li>
		<li>Embed the excised tissue in freezing/mounting medium on dry ice. Add enough medium to cover the gland. When the medium is solidified, transfer the tissues to a freezer at -80 &#176;C for later use9.</li>
		<li>Using a cryomicrotome set at &#8804; -35 &#176;C, cut the tissues into 7-10 &#956;m-thick sections and place them on microscope slides. 
		NOTE: When possible, place two sections on each slide; the left section will be used as a negative control to verify the specificity of the antibodies and the autofluorescence of the tissue, while the right side will be labeled with the antibodies.</li>
		<li>Keep the sections at -80 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Co-IF staining
	<ol>
		<li>Retrieve the appropriate microscopic slides from the freezer and immediately fix the sections by immersing them in formaldehyde 4% for 15 min at room temperature (RT).</li>
		<li>Then immerse the slides in phosphate-buffered saline (PBS) at RT. Leave the slides in PBS at RT until proceeding to the next step.</li>
		<li>Circle each section of the slide using a commercially available hydrophobic barrier or a water-repellent lab pen (see the Table of Materials). Be careful not to touch the tissue. Immediately add drops of PBS to the tissue and place the slide in a humid histology chamber for the remainder of the procedure. 
		NOTE: The tissue sections must remain moisturized. Alternatively, use a box with a lid and place wet paper towels on the bottom.</li>
		<li>Block each tissue section with 100-200 &#181;L of 3% bovine serum albumin (BSA)-Tris-buffered saline (TBS)-0.1% polysorbate 20 (see the Table of Materials) for 30 min at RT. While the samples are blocking, prepare the primary and secondary antibody solutions by diluting the antibodies in TBS-0.1% polysorbate 20. 
		NOTE: The required concentration for the antibody is provided by the manufacturer; see the Table of Materials and Figures 1 and 2 for examples, as well as Dianati et al.9. Although it is not necessary to work in the dark when using most fluorophore-conjugated antibodies, avoid exposing the antibody solutions or stained tissues to intense, bright light.</li>
		<li>Remove the blocking solution by aspiration and incubate the sections in 100-200 &#181;L of the diluted primary antibody for 60 min at RT. Alternatively, incubate with the primary antibody overnight at 4 &#176;C.</li>
		<li>Remove the primary antibody solution by aspiration and wash the sections with 250-500 &#181;L of TBS-0.1% polysorbate 20 for 5 min. Remove the wash solution by aspiration and repeat the wash twice.</li>
		<li>Remove the wash solution by aspiration and incubate the sections with 100-200 &#181;L of the appropriate fluorophore-conjugated secondary antibody for 60 min at RT.</li>
		<li>Remove the secondary antibody solution by aspiration and wash the sections with 250-500 &#181;L of TBS-0.1% polysorbate 20 for 5 min. Remove the wash solution and repeat twice.</li>
		<li>Repeat step 2.5-2.8 using the appropriate combination of primary and secondary antibodies for the subsequent protein(s) of interest.</li>
		<li>Remove the wash solution by aspiration and perform the nuclei staining by incubating the section with 100-200 &#181;L of 1 mg/mL 4&#39;,6-diamidino-2-phenylindole (DAPI) in TBS-0.1% polysorbate 20 for 5 min at RT.</li>
		<li>Remove the DAPI solution by aspiration and mount the slides using a water-soluble, non-fluorescing mounting medium (see the Table of Materials) and coverslips. Proceed one slide at a time. 
		NOTE: Incubating the nucleus stain for more than 5 min will not change the intensity of the staining. Alternatively, remove the DAPI solution on all slides and incubate the tissues in PBS while mounting the slides.</li>
		<li>Place the slides flat in a 4 &#176;C refrigerator for at least 8 h. Proceed to the fluorescence microscopic imaging (see Figures 1 and 2).</li>
	</ol>
	</li>
	<li>Microscopic imaging
	<ol>
		<li>Visualize the fluorophore-conjugated secondary antibodies using a confocal microscope equipped with the various lasers required to excite the fluorophores at their specific wavelengths. 
		Note: To be able to visualize ducts and alveoli, a 40X objective with a numerical aperture of 0.95 is suggested. An example of the specific settings is provided in Figure 1.</li>
		<li>Verify the localization of each protein individually by scanning the image one wavelength at the time. 
		Note: At this stage, it is important to critically analyze the localization of the junctional proteins. To be able to form junctional nexuses, these proteins must be localized at the plasma membrane.</li>
		<li>Determine the co-localization of proteins by merging the images scanned with the lasers at the different wavelengths. 
		Note: Protein co-localization can be visualized by the change of color resulting from the emission of two or more fluorophores at the same location and can be measured using the appropriate software (Figures 1 and 2; also see Reference 9).</li>
	</ol>
	</li>
</ol>
2. Studying PPIs
NOTE: Abdominal mammary glands should be used to study PPIs, as thoracic glands are in close association with the pectoral muscles. Excise the mammary glands (for a complete description of this procedure, refer to Plante et al.)14 and keep them at -80 &#176;C for later use.
<ol>
	<li>Lysate preparation
	<ol>
		<li>Place weighing paper and 2 mL microcentrifuge tubes on dry ice to pre-cool them before proceeding with the next steps.</li>
		<li>Take the mammary gland tissue from -80 &#176;C and keep them on dry ice.</li>
		<li>Weigh the tissues on the pre-chilled weighing papers and then transfer the tissue to 2 mL microcentrifuge tubes (handle on dry ice). Use between 50 and 100 mg of tissue per sample. Keep the tissue on dry ice until step 2.1.5.</li>
		<li>Prepare the required amount of triple detergent lysis buffer supplemented with NaF, NaVO3, and protease/phosphatase inhibitor, as indicated in the Table of Materials, using the following formulas. Mice: required buffer (&#181;L) = mouse tissue weight (mg) x 3; Rat: required buffer (&#181;L) = rat tissue weight (mg) x 5.</li>
		<li>Add the required amount of ice-cold lysis buffer (calculated in step 2.1.4) to the 2 mL tube containing the tissue. 
		NOTE: In steps 2.1.5-2.1.6, proceed with one single tube at a time.</li>
		<li>Homogenize the tissue for 30-40 s using continuous homogenization on a tissue grinder; always keep the tube on ice. Adjust the tissue homogenizer to medium speed and gently move the grinder up and down inside the tube.</li>
		<li>Repeat steps 1.6 and 1.7 with the other tubes.</li>
		<li>Incubate the lysates on ice for 10-30 min.</li>
		<li>Centrifuge the tubes at 170 x g for 10 min at 4 &#176;C.</li>
		<li>Meanwhile, identify 6-10 microcentrifuge tubes (0.6 mL) for each sample and keep them on ice.</li>
		<li>Once the centrifugation is done, check the tubes. Ensure that they contain a top layer of fat, clear, yellow-to-pink lysates (depending on the stage of development) and a pellet.</li>
		<li>Create a hole in the lipid layer using a 200-&#181;L pipette tip to access the liquid phase. Change the tip and collect the lysate without disturbing the pellet or aspirating the lipid layer. Aliquot the lysate in pre-labeled tubes on ice (step 2.1.11) and store them at -80 &#176;C.</li>
		<li>Use an aliquot to quantify the protein concentration using an appropriate commercially available kit (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Indirect immunoprecipitation
	<ol>
		<li>On ice, thaw two aliquots of the total mammary gland lysates prepared previously. 
		NOTE: One aliquot will be used for the IP of the target protein, while the other will serve as the negative control.</li>
		<li>Collect 500-1,000 &#181;g of the lysate and dilute it in PBS to reach a final volume of 200 &#181;L in each 1.5 mL tube. 
		NOTE: The amount of lysate to be used depends on the abundance of the protein of interest and the efficiency of the antibody (see Figure 3 for an example, as well as the Table of Materials). To optimize for each target, different amounts of lysate (i.e., 500, 750, and 1,000 &#181;g) and antibody (i.e., 5, 10, and 20 &#181;g) should be used. Proceed with the following steps (2.2.3-2.3.7.4).</li>
		<li>Add the antibody against the antigen of interest to the first tube of lysate and keep it on ice. 
		NOTE: The required amount is usually suggested on the instruction sheet provided by each company (see the Table of Materials).</li>
		<li>In the second tube, prepare a negative control by adding the same concentration of isotype IgG control as the antibody used in step 2.2.3.</li>
		<li>Incubate the tubes overnight at 4 &#176;C on a tube roller-mixer at low speed.</li>
		<li>The following day, add 50 &#181;L of magnetic beads to new 1.5 mL tubes for pre-washing.
		<ol>
			<li>Select either Protein A or Protein G magnetic beads based on the relative affinity to the antibody.</li>
			<li>It is important to avoid using aggregated beads; gently mix the bead suspension until it is uniformly re-suspended before adding it to the tubes.</li>
		</ol>
		</li>
		<li>Place the tubes containing the beads on the magnetic stand and allow the beads to migrate to the magnet. Remove the storage buffer from the beads using a 200 &#181;L pipette.</li>
		<li>Wash the beads by adding 500 &#181;L of PBS-0.1% polysorbate 20 and vortex the tubes vigorously for 10 s.</li>
		<li>Put the tubes back on to the magnetic stand and allow the beads to migrate to the magnet.</li>
		<li>Remove the excess wash buffer by pipetting with a 200 &#181;L pipette.</li>
		<li>Add the reaction complex (lysate-antibody) from step 2.2.5 to the beads and incubate for 90 min at RT on the roller mixer.</li>
		<li>Place the tubes on the magnetic stand and allow the beads to migrate to the magnet. Using a 200 &#181;L pipette, aspirate and discard the lysate and place the tubes on ice.</li>
		<li>Wash the beads by adding 500 &#181;L of PBS, placing the tubes on the magnetic stand, and removing the liquid using a 200 &#181;L pipette. Repeat this wash step. During the wash steps, avoid vortexing and keep the samples on ice.</li>
		<li>Wash the beads once with PBS-0.1% polysorbate 20 without vortexing and discard the last wash buffer using a 200 &#181;L pipette tip.</li>
		<li>To elute, add 20 &#181;L of 0.2 M acidic glycine (pH = 2.5) to the tubes and shake them for 7 min on the roller mixer.</li>
		<li>Centrifuge at high speed for a few seconds (quick spin) and collect the supernatant in a fresh ice-cold tube.</li>
		<li>Repeat steps 2.2.14 and 2.2.15 for each tube. 
		NOTE: The final volume will be 40 &#181;L.</li>
		<li>Add 10 &#181;L of 4x Laemmli buffer to the 40-&#181;L eluted sample from step 2.2.16. 
		NOTE: The color will turn yellow due to the acidic pH.</li>
		<li>Immediately add 1 M Tris (pH = 8), one drop at a time, to the eluted sample from step 2.2.18 until its color turns blue. Proceed to the next tubes.</li>
		<li>Boil the samples from step 2.2.18 at 70-90 &#176;C for 10 min. Proceed immediately to gel electrophoresis. Alternatively, transfer the samples to a freezer at -80 &#176;C until loading.</li>
	</ol>
	</li>
	<li>Downstream application: gel electrophoresis followed by Western blot
	<ol>
		<li>Prepare separating and stacking SDS-PAGE acrylamide gels (1.5 mm thickness) following standard procedures15. 
		NOTE: The choice of gel (8-15% acrylamide, gradient: see the Table of Materials) should be determined based on the molecular size of the protein to be precipitated and of the potential binding partners to be analyzed. These proteins must be resolved from each other to allow for proper immunodetection.</li>
		<li>Thaw the immunoprecipitation (IP)-eluted samples (step 2.2.20) on ice.</li>
		<li>Prepare protein lysates from the same samples (used for the IP procedure above). Use 50 &#181;g of total lysate and add 4X Laemmli sample buffer. Boil the samples at 70-90 &#176;C for 5 min and place on ice until loading. 
		NOTE: These samples will be loaded beside the eluted IP sample to demonstrate the presence of precipitated proteins in the total lysate.</li>
		<li>Load the prepared lysates from step 2.3.3 and the precipitated samples from step 2.2.20 side-by-side in an acrylamide gel and run them in running buffer (10x running buffer: 30.3 g of Tris, 144.1 g of glycine, and 10 g of SDS in 1 L of distilled water) at 100 V for approximatively 95 min, or until the edge of the migrating proteins reaches the bottom of the gel.</li>
		<li>Transfer the gels to a nitrocellulose or PVDF membrane using a standard protocol9,15.</li>
		<li>Block the membrane for 1 h on a rocker on low speed in 5% dry milk-TTBS (20 mM Tris, 500 mM NaCl, and 0.05% polysorbate 20).</li>
		<li>Identify whether the precipitation was successful.
		<ol>
			<li>Probe the membrane using the first antibody against the precipitated protein, diluted in 5% dry-milk-TTBS at the concentration recommended by the manufacturer, overnight at 4 &#176;C on a rocking platform with slow agitation. 
			NOTE: See the Table of Materials for recommendations.</li>
			<li>The following day, wash the membrane 3 times for 5 min each with TTBS on a rocking platform with high agitation.</li>
			<li>Incubate the membrane in the appropriate secondary antibody conjugated with horseradish peroxidase (HRP), diluted in TTBS, for 1 h at RT on a rocking platform with slow agitation. 
			NOTE: Alternatively, a secondary antibody conjugated with a fluorochrome can be used if an appropriate apparatus to detect the signal is available.</li>
			<li>Perform 3 to 6 washes, each for 5 min, with TTBS on a rocking platform with high agitation. Analyze the signal of the secondary antibody by incubating the membrane with a commercially available luminol solution (see the Table of Materials) and follow the manufacturer instructions. Detect the signal using a chemiluminescence imaging system (see the Table of Materials). 
			NOTE: For a detailed protocol on Western blot analysis, see Reference 16.</li>
		</ol>
		</li>
		<li>To identify interacting proteins, perform steps 2.3.7.1-2.3.7.4 using the appropriate antibodies on the same blot. 
		NOTE: If proteins are interacting, the binding partners will be co-immunoprecipitated with the target protein and will thus be detectable by Western blotting. Step 2.3.8 can be repeated with more antibodies to determine whether other proteins reside in the same proteins complex, as long as the molecular weights of the proteins differ enough to be well-separated on the gel and membrane.</li>
		<li>To confirm that the identified binding partners are not artifacts, reciprocal IP should be performed. 
		NOTE: This is performed by repeating steps 2.2.1-2.2.20 with the same lysate but precipitating one of the binding partners identified in step 3.8. Then, steps 3.1-3.8 are repeated using the primary antibody against the first protein of interest.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to declare.

Cell-cell interactions via junctions are required for the proper function and development of many organs, such as the mammary gland. Studies have shown that junctional proteins can regulate the function and stability of one another and activate signal transduction by tethering each other at the cell membrane10. The protocols presented in the current manuscript have provided interesting findings about junctional protein differential expression, localization, and interaction during normal murine gland development9. Given that junctional protein localization is critical to the function of the proteins, and because they are known to interact with scaffolding proteins and numerous kinases3, co-IF and co-IP are effective techniques in the field of cell-cell interactions. Not only are these methods essential to enlighten the necessity of junctional nexuses in mammary gland development and their dysregulation in breast cancer, but they can also be used in other tissues and in experiments using cell lines.
The mammary gland is composed of two main compartments: the stroma and epithelium4. The adult epithelium is made of two layers of cells. In this approach, the proximity of the potential binding partners was determined using the co-IF technique, and their physical interactions were confirmed using co-IP. Co-IF has been successfully used by others to demonstrate the co-localization of proteins within the same tissue, structure, cell, or intracellular compartment17,18. The main advantage of this technique is the visual information it brings about the cellular or subcellular localization of each protein within the different cell types composing the tissue. Although this technique is quite simple, some recommendations must be followed. For instance, to avoid tissues damage, always add the solutions one drop at a time using a 200 &#181;L pipette or a transfer pipette. This will allow for the immersion of the tissue surface without damaging the tissues. Similarly, remove the solutions using a Pasteur pipette placed beside the tissues by gently tilting the slide. Moreover, for antibodies whose storage solution contains glycerol, careful suction of the wash buffers is required to reduce the background. Moreover, the presence of milk proteins, such as caseins, during lactation can interfere with the antibodies by trapping them, resulting in false positives. A critical analysis of the resulting image is thus required, specifically at this stage.
This co-IF technique also has certain limitations or pitfalls. First, it requires specific antibodies. As mentioned previously (step 1.1), it is recommended to use one section (i.e., the one on the right side) on each slide for staining following the steps described above, adding the primary and secondary antibodies sequentially. For the remaining section (i.e., the one on the left side), follow the same procedure, using TBS-polysorbate 20 0.1% instead of the primary antibody solutions. While it is also possible, and even better, to verify the specific binding of the antibody using peptide competition, peptides used to generate commercial antibodies are not always available. It is thus important to verify the specificity of the binding using positive and negative controls (i.e., tissues or cells known to express, or not, the protein of interest). Moreover, antigen fixation sites can be inaccessible, especially for formalin-fixed tissues, thus resulting in the absence of a specific signal. An antigen-retrieval procedure may thus be required for some tissues. A short incubation with detergent can also be performed prior to antigen-retrieval to permeabilize the cell membrane. Second, for multiplexing, antibodies must be raised in different animals. For instance, if anti-rabbit was used in step 1.2.6, anti-mouse could be selected in step 1.2.10, but not another antibody raised in rabbit. Since most commercial antibodies are raised in rabbits, mice, or goats, it is sometimes difficult, or even impossible, to target two proteins at the same time due to the lack of appropriate antibodies. To overcome these limitations, one can either buy commercially available, pre-labeled antibodies or label primary antibodies with fluorophores using commercially available kits. Third, another shortcoming is linked to the excitation and emission of the different fluorophores. To avoid overlap of the signals from two different antibodies, the excitation-emission spectrum of each fluorophore must be separated. Thus, the number of targets that can be analyzed at once will vary according to the configuration of the available microscope. Finally, the quality of the analysis is highly dependent upon the microscope used. More detailed and precise data can be obtained using a confocal microscope compared to an epifluorescent microscope. The use of super-resolution microscopy can reveal protein co-localization in even more detail19.
Although co-IF brings important insights about the proximity of potential binding partners, it should be complemented by other methods to identify physical interactions between proteins. Among the available methods, co-IP is probably one of the most affordable to perform, as the equipment and material are easily accessible. Using an antibody bound to magnetic beads, one can isolate protein complexes and identify the components present in that complex using typical Western blot analysis. Similar to co-IF, some recommendations should be followed for best practices. For instance, it is recommended to minimize the samples when homogenizing the tissues to reduce the time between steps 2.1.5 and 2.1.9. While an experienced person can process up to 10 samples at a time, a beginner should not handle more than 4-6 samples. Similarly, the number of tubes should be limited when performing the IP protocol for the first time. It is recommended to start with a negative control (i.e., IgG) and a positive control (i.e., a tissue known to contain the protein to be immunoprecipitated) only. The second trial should be dedicated to optimization (see step 2.2.2). Once these steps give satisfying results, samples to be analyzed can be processed. Note that a negative control should always be included in the procedure.
This co-IP technique also has potential pitfalls. First, it requires tissues to be homogenized in conditions permitting the preservation of the links between proteins. For membrane proteins, such as junctional proteins, it is also crucial to use a buffer that will preserve the bonds between the proteins while also allowing their solubility. Second, similar to co-IF, it requires high-affinity antibodies for both the target protein and the binding partners. Moreover, because proteins remain in their tertiary conformation and protein complexes are not dissociated by homogenization, if the antibody recognizes part of the target protein that is in close proximity to the binding domain of a partner or that is hidden inside the native structure of the protein, the IP can be compromised. It is thus essential to always verify the efficiency of the IP using Western blotting before concluding the absence of a binding partner. Third, co-IP can generate false-positive results because of the protein either binding directly to the beads or precipitating during the procedure without being part of the complex. To identify these artifacts, an IgG control is required, as well as a reciprocal IP, as described in the methods presented. It is also possible to add a &#34;pre-cleaning&#34; procedure between steps 2.2.2 and 2.2.3 to avoid the unspecific binding of proteins to the beads. To do so, incubate the lysates with 50 &#181;g of beads for 1 h at 4 &#176;C on a roller mixer. Remove the beads with the magnetic stand and proceed to step 2.2.3. Fourth, the heavy and light chains of IgG can be the same size as the proteins of interest or the binding partner, thereby masking the signal. One solution is to dissociate the IgG chains with glycine, as described in this manuscript. It is also possible to purchase secondary antibodies that only recognize native antibodies and therefore do not bind to the denatured light and heavy chains loaded in the membrane (see the Table of Materials). The two methods may sometimes have to be combined. Fifth, co-IP allows for the identification of a limited number of binding partners, in part because of the number of antibodies that can be probed on the membrane. It also requires the pre-identification of these binding partners, either by co-IF or through a literature review. Finally, co-IP allows for the identification of the proteins present within a complex, and not for the direct interaction between two proteins.
Results from co-IP can be analyzed in different ways. It is possible to solely identify interacting partners by re-probing the same membrane with different antibodies, as described in this manuscript. It is also possible to quantify this interaction. To do so, the amount of the protein immunoprecipitated is first quantified by probing the membrane with an antibody against this protein. The signal intensity is analyzed using an imaging software, as described in this manuscript. Then, the membrane is re-probed with an antibody against the binding partner, and the signal intensity also quantified. The interactions between the two proteins can then be expressed as a ratio of the amount of the binding partner to the amount of the immunoprecipitated protein. However, to allow for comparison, the different samples must be processed at the same time and loaded on the same membrane. Biological replicates can be proceeded and analyzed similarly, and statistical analysis can be performed.
In the last few years, other techniques were developed to analyze PPIs. For instance, FRET allows for the identification of interacting proteins through energy transfer from one tag to another, only when the proteins are close enough to interact20. However, because it requires proteins to be tagged, this technique cannot be used in tissues. Similarly, it is possible to identify PPIs using a protein fused with a bacterial biotin ligase, BirA21. This ligase will add biotin to proteins that come in close proximity (i.e., interact) with the chimeric protein. While this method is innovative and unbiased, it cannot be performed in tissues. Alternatively, PLA can be used in tissues. Similar to co-IF, this assay is based on antibody affinity. For this assay, secondary antibodies are tagged with DNA sequences that can interact when they are in close proximity (i.e., upon PPIs) and form a circular DNA molecule22. This circular DNA molecule is then amplified and detected using fluorescently labeled complementary oligonucleotides. Although this assay is elegant, it requires many validation steps and still relies on primary antibody affinity and some knowledge of potential interacting partners. Finally, an unbiased alternative to the traditional IP assay has also been developed to identify PPIs. In rapid immunoprecipitation-mass spectrometry of endogenous protein (RIME) assays, IP samples are analyzed by mass spectrometry (IP/MS)23 instead of Western blot analysis. The main advantage of this high-throughput method is that it provides massive information about all the endogenous interacting proteins using few materials. However, it requires access to a peptide-sequencing instrument23.
It is important to mention that, after several preliminary tests, each step of this protocol has been optimized for the mammary gland. However, the method can surely be used for other organs after a few modifications. For co-IF, the optimal temperature for mammary gland sectioning, suitable blocking and washing and solutions, and the proper antibody concentrations were all tested. For co-IP, various lysis and elution buffers and methods of extractions were also tested and have a major impact on IP success. In sum, each step of this protocol is important to obtaining high-quality and reproducible results with the least possible background and the most specificity. While other methods are available, co-IF followed by co-IP remain valid and simple methods to evaluate PPIs. These two techniques can be used both in tissues and in cell lines and only require a few validation steps and controls.

Mammary gland growth and development occurs mainly after birth. This organ constantly remodels itself throughout the reproductive life of a mammal1. The adult mammary gland epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of basal cells, mainly composed of myoepithelial cells, surrounded by a basement membrane2. For a good review on mammary gland structure and development, the reader can refer to Sternlicht1. Cell-cell interactions via gap (GJ), tight (TJ), and adherens (AJ) junctions are necessary for the normal development and function of the gland1,3,4,5,6. The main components of these junctions in the murine mammary gland are Cx26, Cx30, Cx32, and Cx43 (GJ); Claudin-1, -3, -4, and -7 and ZO-1 (TJ); and E-cadherin, P-cadherin, and &#946;-catenin (AJ)7,8. The levels of expression of these different junctional proteins vary in a stage-dependent manner during mammary gland development, suggesting differential cell-cell interaction requirements9. GJ, TJ, and AJ are linked structurally and functionally and tether other structural or regulatory proteins to the neighboring sites of adjacent cells, thus creating a junctional nexus10. The composition of the junctional nexus can impact bridging with the underlying cytoskeleton, as well as nexus permeability and stability, and can consequently influence the function of the gland8,9,10,11. The components of intercellular junctions residing in junctional nexuses or interacting with one another at different developmental stages of mammary gland development were analyzed recently using co-immunofluorescence (co-IF) and co-immunoprecipitation (co-IP)9. While other techniques allow for the evaluation of the functional association between proteins, these methods are not presented in this manuscript.
As proteins merely act alone to function, studying protein-protein interactions (PPIs), such as signal transductions and biochemical cascades, is essential to many researchers and can provide significant information about the function of proteins. Co-IF and microscopic analysis help to evaluate a few proteins that share the same subcellular space. However, the number of targets is limited by the antibodies, which must be raised in different animals, and by the access to a confocal microscope equipped with different wavelength lasers and a spectral detector for multiplexing. Co-IP confirms or reveals high-affinity physical interactions between two or more proteins residing within a protein complex. Despite the development of novel techniques, such as fluorescence resonance energy transfer (FRET)12 and proximity ligation assay (PLA)13, which can simultaneously detect the localization and interactions of proteins, co-IP remains an appropriate and affordable technique to study interactions between endogenous proteins.
The step-by-step method described in this manuscript will facilitate the study of protein localization and PPIs and point out pitfalls to avoid when studying endogenous PPIs in the mammary glands. The methodology starts with the presentation of the different preservation procedures for the tissues required for each technique. Part 1 presents how to study protein co-localization in three steps: i) the sectioning of mammary glands, ii) the double- or triple-labeling of different proteins using the co-IF technique, and iii) the imaging of protein localization. Part 2 shows how to precipitate an endogenous protein and identify its interacting proteins in three steps: i) lysate preparation, ii) indirect protein immunoprecipitation, and iii) binding partner identification by SDS-PAGE and Western blot. Each step of this protocol is optimized for rodent mammary gland tissues and generates high-quality, specific, and reproducible results. This protocol can also be used as a starting point for PPI studies in other tissues or cell lines.

<table>
	<tbody>
		<tr>
			<td>Mice strain and stage&#160;</td>
			<td>St. Constant, Quebec, Canada</td>
			<td></td>
			<td>C57BL/6 Femals; pregnancy day 18 (P18) and lactation day 14 (L14), Charles River Canada&#160;</td>
		</tr>
		<tr>
			<td>PBS 10X (stock)</td>
			<td></td>
			<td></td>
			<td>1)&#160;&#160;&#160;&#160;&#160; Dissolve 80g NaCl (F.W.: 58.44), 2g KCl (F.W. 74.55), 26.8g Na2HPO4&#8226;7H2O (F.W. 268.07) and 2.4g KH2PO4 (F.W.:136.09) in 800ml distilled water.&#160; 
			2)&#160;&#160;&#160;&#160;&#160; Adjust the PH to 7.4&#160; 
			3)&#160;&#160;&#160;&#160;&#160; Add water to reach to the 1 litre final volume.&#160;</td>
		</tr>
		<tr>
			<td>TBS 10X (stock)</td>
			<td></td>
			<td></td>
			<td>1)&#160;&#160;&#160;&#160;&#160; Dissolve 60.5g TRIS, 87.6g NaCl in 800ml distilled water.&#160; 
			2)&#160;&#160;&#160;&#160;&#160; Adjust the PH to 7.5&#160; 
			3)&#160;&#160;&#160;&#160;&#160; Add water to reach to the 1 litre final volume.&#160;</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Part 1-Immunofluorescence</td>
		</tr>
		<tr>
			<td>Freezing media</td>
			<td>VWR International, Ville Mont-Royal, QC, Canada</td>
			<td>95067-840</td>
			<td>VMR frozen sections compound&#160;</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Mississauga, ON, Canada</td>
			<td>956640</td>
			<td>Microm HM525, Thermo fisher scientific HM525 NX Cryostat 115V 60Hz</td>
		</tr>
		<tr>
			<td>Blades</td>
			<td>C.L. Sturkey, Inc. Les Produits&#160;Scientifiques&#160;ESBE&#160;St-Laurent, QC, Canada</td>
			<td>&#160;BLM1001C</td>
			<td>High profile gold coated blades</td>
		</tr>
		<tr>
			<td>Pap pen</td>
			<td>Cedarlane, Burlington, ON, Canada</td>
			<td>8899</td>
			<td>Super PAP Pen, Thermo fisher scientific</td>
		</tr>
		<tr>
			<td>Microscopic slides</td>
			<td>Fisher Scientific, Burlington, ON, Canada</td>
			<td>12-550-15</td>
			<td>Fisherbrand&#160;Superfrost Plus Microscope Slides</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>BioShop Canada Inc, Burlington, ON, Canada</td>
			<td>FOR201.1</td>
			<td>Forlmadehyde</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td></td>
			<td></td>
			<td>Santa Cruz Biotechnology, Inc, California, USA</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>3% BSA in TBS</td>
		</tr>
		<tr>
			<td>Wash solution</td>
			<td></td>
			<td></td>
			<td>TBS-Tween 20 0.1%</td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Oakville, ON, Canada</td>
			<td>P 9416</td>
			<td>Tween 20, Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Cedarlane, Burlington, ON</td>
			<td>17984-25(EM)</td>
			<td>Fluoromount-G</td>
		</tr>
		<tr>
			<td>First &#38; secondary antibodies&#160;</td>
			<td>Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in Column D</td>
			<td>E-Cadherin (4A2) Mouse mAb (#14472s) 1/50 (Cell Signaling) with anti-mouse IgG Fab2 Alexa Fluor 555 (#4409s), Cell Signaling&#160;</td>
		</tr>
		<tr>
			<td>First &#38; secondary antibodies&#160;</td>
			<td>Life technologies, Waltham, MA, USA &#38; Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in Column D</td>
			<td>Claudin-7 (#34-9100) 1/100 (Life Technologies) with anti-rabbit IgG Fab2 Alexa Fluor 488 (#4412s) (Cell Signaling)&#160;</td>
		</tr>
		<tr>
			<td>First &#38; secondary antibodies&#160;</td>
			<td>Santa Cruz Biotechnology, Inc, California, USA; Fischer Scientific, Burlington, ON, Canada&#160;</td>
			<td>Mentioned in Column D</td>
			<td>&#946;-Catenin Antibody (C-18): sc-1496 (SANTA CRUZ) with anti-Goat IgG (H+L) Alexa Fluor 568 (#A11057), Molecular Probe (Fisher Scientific)</td>
		</tr>
		<tr>
			<td>First &#38; secondary antibodies&#160;</td>
			<td>Life technologies, Waltham, MA, USA &#38; Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in Column D</td>
			<td>Connexin26&#160; (#33-5800) 1/75 (Life Technologies) with anti-mouse IgG Fab2 Alexa Fluor 647 (#4410s)&#160;</td>
		</tr>
		<tr>
			<td>Nuclei stain</td>
			<td>Fisher Scientific, Burlington, ON, Canada</td>
			<td>D1306</td>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride) 1/1000 in PBS</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon Canada, Mississauga, On, Canada</td>
			<td></td>
			<td>Nikon A1R+ confocal microscopic laser equipped with a spectral detector&#160;</td>
		</tr>
		<tr>
			<td>Software of IF images analysis</td>
			<td>Nikon Canada, Mississauga, On, Canada</td>
			<td></td>
			<td>NIS-elements software (version 4)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Part 2-Immunoprecipitation</td>
		</tr>
		<tr>
			<td>Triple-detergent Lysis buffer (100ml) pH=8.0&#160;</td>
			<td></td>
			<td></td>
			<td>1) Mix 50mM TRIS (F.W.&#160;:121.14), 150mM NaCl (F.W.&#160;:58.44), 0.02% Sodium Azide, 0.1% SDS, 1% NONIdET P40, 0.5% Sodium Deoxycholate in 80ml distilled H2O.&#160; 
			2) Adjust the PH to 8.0 with HCl 6N (~0.5ml). 
			3) Adjust the volume to 100ml. Keep it in fridge.&#160; 
			At the day of protein extraction, use 1/100 NaVo3, 1/100 protease/phosphatase inhibitor and 1/25 NAF in calculated amount of Triple detergent lysis buffer: 
			Sodium Fluorid (stock) solution 1.25M (F.W. 41.98), Sodium Orthovanadate (stock) Solution 1M (F.W.: 183.9)</td>
		</tr>
		<tr>
			<td>Protease/phosphatase inhibitor</td>
			<td>Fisher Scientific, Burlington, ON</td>
			<td>78441</td>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X)</td>
		</tr>
		<tr>
			<td>Protein dosage</td>
			<td>Thermo Scientific, Rockford, Illinois, USA</td>
			<td>23225</td>
			<td>Pierce BCA protein assay kit&#160;</td>
		</tr>
		<tr>
			<td>Tissue grinder</td>
			<td>Fisher Scientific, Burlington, ON</td>
			<td>FTH-115</td>
			<td>Power 125, Model FTH-115</td>
		</tr>
		<tr>
			<td>Magnetic beads and stand</td>
			<td>Millipore, Etobicoke, ON, Canada</td>
			<td></td>
			<td>PureProteome Protein G Magnetic Bead System (LSKMAGG02)</td>
		</tr>
		<tr>
			<td>Wash solution for IP</td>
			<td></td>
			<td></td>
			<td>PBS or PBS-Tween20 0.1% depending to the step</td>
		</tr>
		<tr>
			<td>Primary antibodies for immunoprecipitation</td>
			<td>Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in coulmn D</td>
			<td>IgG Rabbit (rabbit (DA1E) mAb IgG Isotype control (#3900s) (Cell Signaling) 0.5 &#181;l/200 &#181;l</td>
		</tr>
		<tr>
			<td>Primary antibodies for immunoprecipitation</td>
			<td>Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in coulmn D</td>
			<td>IgG Mouse mouse (G3A1) mAb IgG Isotype control (#5415s) (Cell Signaling) 0.5 &#181;l/200 &#181;l&#160;</td>
		</tr>
		<tr>
			<td>Primary antibodies for immunoprecipitation</td>
			<td>Sigma-Aldrich, Oakville, ON, Canada</td>
			<td>Mentioned in coulmn D</td>
			<td>Connexin43 (#C6219) (Sigma-Aldrich) 4 &#181;l/200 &#181;l&#160;</td>
		</tr>
		<tr>
			<td>Primary antibodies for immunoprecipitation</td>
			<td>Cell Signaling, Beverly, MA, USA</td>
			<td>Mentioned in coulmn D</td>
			<td>E-cadherin (4A2) Mouse mAb (#14472s) (Cell Signaling) 1 &#181;l/200 &#181;l&#160;</td>
		</tr>
		<tr>
			<td>Laemmli buffer&#160;</td>
			<td>BIO-RAD, Mississauga, Ontario, Canada</td>
			<td>1610747</td>
			<td>4x Laemmli Sample Buffer (Add &#946;-mercaptoethanol following manufacturer recommendation)</td>
		</tr>
		<tr>
			<td>Acidic glycine&#160;</td>
			<td>Fisher Scientific, Burlington, ON</td>
			<td>PB381-5</td>
			<td>0.2 M glycine; adjust pH=2.5 with HCl&#160;</td>
		</tr>
		<tr>
			<td>Tris&#160;</td>
			<td>Fisher Scientific, Burlington, ON</td>
			<td>BP152-1</td>
			<td>1 M (pH=8)&#160;</td>
		</tr>
		<tr>
			<td>SDS-PAGE acrylamide gels&#160;</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1610180 -5</td>
			<td>TGX Stain-Free FastCast Acrylamide Solutionss (7.8%, 10%, 12%)</td>
		</tr>
		<tr>
			<td>Running buffer 10x&#160;</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1704272</td>
			<td>Tris 30.3g/glycine 144.1g /SDS 10g in 1 litre distilled water</td>
		</tr>
		<tr>
			<td>Membranes</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1704272</td>
			<td>PVDF membranes, Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td>
		</tr>
		<tr>
			<td>Transfer method</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1704155</td>
			<td>Trans-Blot Turbo Transfer System</td>
		</tr>
		<tr>
			<td>Dry Milk</td>
			<td>Smucker Food of Canada Co, Markham, ON, Canada</td>
			<td></td>
			<td>Fat Free Instant Skim Milk Powder, Carnation</td>
		</tr>
		<tr>
			<td>Blocking solution for blots</td>
			<td></td>
			<td></td>
			<td>5% dry milk in TBS-Tween 20 0.1%</td>
		</tr>
		<tr>
			<td>Washing solutions for blots</td>
			<td></td>
			<td></td>
			<td>TBS-Tween 20 0.1%</td>
		</tr>
		<tr>
			<td>Primary and secondary antibodies for blots (10ml)</td>
			<td>Sigma-Aldrich, Oakville, Ontario &#38; Abcam, Toronto, ON, Canada</td>
			<td>Mentioned in column D</td>
			<td>Connexin43 (#C6219) (Sigma-Aldrich) 1/2500 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5000 (Abcam, Toronto, ON, Canada)</td>
		</tr>
		<tr>
			<td>Primary and secondary antibodies for blots (10ml)</td>
			<td>Cell Signaling, Beverly, MA, USA &#38; Abcam, Toronto, ON, Canada</td>
			<td>Mentioned in column D</td>
			<td>E-cadherin (24E10) rabbit mAb 1/1000 (#3195s) (Cell Signaling) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody&#160; (ab131366) 1/5000 (Abcam, Toronto, ON, Canada)</td>
		</tr>
		<tr>
			<td>Primary and secondary antibodies for blots (10ml)</td>
			<td>Life technologies, Waltham, MA, USA &#38; Abcam, Toronto, ON, Canada</td>
			<td>Mentioned in column D</td>
			<td>Claudin-7 (#34-9100) (Life technologies) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366)&#160; 1/5000 (Abcam, Toronto, ON, Canada)</td>
		</tr>
		<tr>
			<td>Primary and secondary antibodies for blots (10ml)</td>
			<td>Life technologies, Waltham, MA, USA &#38; Abcam, Toronto, ON, Canada</td>
			<td>Mentioned in column D</td>
			<td>Claudin3&#160; (#34-1700) (Life technologies) 1/1000 with HRP-conjugated Veriblot for IP secondary antibody&#160; (ab131366) 1/5000 (Abcam, Toronto, ON, Canada)</td>
		</tr>
		<tr>
			<td>Luminol solution for signal detection on blots</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1705061&#160;</td>
			<td>Clarity Western ECL Blotting Substrate&#160;</td>
		</tr>
		<tr>
			<td>Imaging blots</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td>1708280</td>
			<td>ChemiDoc MP imaging system</td>
		</tr>
		<tr>
			<td>Analayzing blots</td>
			<td>BIO-RAD, Mississauga, ON, Canada</td>
			<td></td>
			<td>ImageLab 5.2 software&#160;</td>
		</tr>
	</tbody>
</table>

analysis of protein-protein interactions and co-localization between components of gap, tight, and adherens junctions in murine mammary glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

To determine whether GJ, AJ, and TJ components can interact together in the mammary gland, co-IF assays were first performed. Cx26, a GJ protein, and &#946;-Catenin, an AJ protein, were probed with specific antibodies and revealed using fluorophore-conjugated mouse-647 (green, pseudocolor) and goat-568 (red) antibodies, respectively (Figure 1B and C). Data showed that they co-localize at the cell membrane of epithelial cells in the mice mammary gland on lactation day 7 (L7), as reflected by the yellow color (Figure 1D). Secondly, Claudin-7, a TJ protein; E-cadherin, an AJ protein; and Connexin26 (Cx26), a GJ protein, were probed with their specific antibodies and were revealed with fluorophore-conjugated rabbit-488 (green), mouse-555 (red), and mouse-647 (coral blue; pseudocolor) antibodies, respectively (Figure 2B-D). E-cadherin and Claudin-7 co-localization is displayed as a yellow-to-light-orange color, while Cx26 co-localization with E-cadherin and Claudin-7 resulted in white punctuated staining in mice mammary glands on pregnancy day 18 (P18) (Figure 2E).
To find out which junctional proteins intermingle and physically tether together at the cell membrane, co-immunoprecipitation was performed using mammary gland tissues from lactating mice (L14). Results showed that Cx43, a component of GJ, interacts with E-Cadherin and Claudin-7, but not with Claudin-3 (Figure 3A and B). These results were confirmed by the reciprocal IP; when E-Cadherin was immunoprecipitated, it interacted with Cx43 and Claudin-7 (Figure 3C).
<img alt="Figure 1" src="/files/ftp_upload/55772/55772fig1.jpg" /> 
Figure 1: &#946;-Catenin and Cx26 co-localize at the cell membrane in mice mammary glands. Cryosections from mammary glands of mice at lactation day 7 (L7) were cut (7 &#181;m) and processed for immunofluorescent staining. (A) Nuclei were stained with DAPI (blue). (B) Cx26 (green, pseudocolor) and (C) &#946;-Catenin (red) are shown combined with appropriate fluorophore-labeled antibodies. (D) A merged image. Images were obtained with a confocal microscope equipped with a spectral detector. DAPI was visualized using the following settings: emission wavelength, 450.0 nm; excitation wavelength, 402.9 nm; laser power, 1.2; detector gain, PMT HV 100; PMT offset, 0. Cx26 (647) was visualized using the following settings: emission wavelength, 700.0 nm; excitation wavelength, 637.8 nm; laser power, 2.1; detector gain, PMT HV 110; PMT offset, 0. &#946;-Catenin (568) was visualized using the following settings: emission wavelength, 595.0 nm; excitation wavelength, 561.6 nm; laser power, 2.1; detector gain, PMT HV 110; PMT offset, 0. Scale bars = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55772/55772fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55772/55772fig2.jpg" /> 
Figure 2: Connexin26 (Cx26), E-cadherin, and Claudin-7 co-localize at the cell membrane in mice mammary glands. Cryosections from mammary glands on pregnancy day 18 (P18) were cut (7 &#181;m) and processed for immunofluorescent staining using (B) Claudin-7 (green), (C) E-Cadherin (red), and (D) Cx26 (coral blue; pseudocolor), combined with the appropriate fluorophore-labeled antibodies. (A) Nuclei were stained with DAPI (blue). Images were obtained with a confocal microscope equipped with a spectral detector. DAPI was visualized using the following settings: emission wavelength, 450.0 nm; excitation wavelength, 402.9 nm; laser power, 5.4; detector gain, PMT HV 65; PMT offset, 0. Claudin-7 (488) was visualized using the following settings: emission wavelength, 525.0 nm; excitation wavelength, 489.1 nm; laser power, 5.0; detector gain, PMT HV 12; PMT offset, 0. E-Cadherin (568) was visualized using the following settings: emission wavelength, 595.0 nm; excitation wavelength, 561.6 nm; laser power, 13.5; detector gain, PMT HV 45; PMT offset, 0. Cx26 (647) was visualized using the following settings: emission wavelength, 700.0; excitation wavelength, 637.8; laser power, 5.0; detector gain, PMT HV 55; PMT offset, 0. Scale bars = 50 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55772/55772fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55772/55772fig3.jpg" /> 
Figure 3: Cx43, E-Cadherin, and Claudin-7, but not Claudin-3, are involved in a protein complex. Cx43 (A and B) and E-Cadherin (C) were immunoprecipitated using 500 mg of total lysates from mammary glands of mice at lactation. IPs and lysates were loaded in gels and transferred on PVDF membranes. Because Claudin-7 and Claudin-3 have the same molecular weight, they could not be analyzed on the same membrane. Thus, two parallel IPs were performed with the same lysate for Cx43, loaded on two gels, and transferred (membranes A and B). Membrane A was first probed with Cx43 to confirm the efficiency of the IP (top panel). Then, the membrane was sequentially probed with E-Cadherin and Claudin-7. Western blot analysis showed that the two proteins interact with Cx43. Membrane B was first probed with Cx43 to confirm the efficiency of the IP (top panel) and then probed with Claudin-3. Western blot analysis showed that Claudin-3 did not IP with Cx43, demonstrating that the two proteins do not interact. Membrane C was first probed with E-cadherin to confirm the efficiency of the IP (top panel). Then, the membrane, was sequentially probed with Cx43 and Claudin-7. Western blot analysis confirmed the interactions between the proteins. <a href="http://cloudflare.jove.com/files/ftp_upload/55772/55772fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands at JoVE.com

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

analysis-protein-protein-interactions-co-localization-between

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.0K Views.  INRS, Institut Armand-Frappier. Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages. 

Video: Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...