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EoE Sub Articles

1. Body Wall Preparation
NOTE: Preparation of third instar larval body walls (for study of the NMJs which innervate the body wall muscles) was performed as previously described but with some modifications.
<ol>
	<li>Dissection
	<ol>
		<li>Raise fly stocks and crosses at 25 &#176;C for five to six days using standard procedures.</li>
		<li>Pick crawling third instar larvae from vials or bottles using fine forceps.</li>
		<li>Wash the larvae in a small Petri dish containing Phosphate Buffer Saline (PBS) to remove any food particles.</li>
		<li>Place a single larva onto a sylgard disc and immerse it in a few drops of ice-cold PBS. Using ice-cold PBS will help stun the larva making it easier to manipulate. Throughout the dissection, ensure that the preparation is always submerged in PBS to prevent it from drying.</li>
		<li>Position the larva with its dorsal side facing up so that the two tracheal tracts are visible under a dissecting microscope (Figure 1A). Using the forceps to grasp a minutien pin, pin the larva down at the posterior end near the spiracles (Figure 1B). With another pin, pierce through the cuticle at the anterior end near the mouth hooks. Gently stretch the larva out lengthwise, then pin it down (Figure 1B).</li>
		<li>Using microdissection scissors, pinch the posterior end near the pin to create a small opening. The incision should be superficial enough to just pass through the cuticle.</li>
		<li>Placing the tip of the bottom blade of the scissors into the incision, cut along the entire length of the dorsal midline between the two tracheal tracts (Figure 1C). Point the scissor blades slightly upwards when cutting to avoid damaging the ventral body wall muscles.</li>
		<li>Make a small horizontal incision slightly anterior to the posteriorly placed pin (Figure 1C). Make another similar incision slightly posterior to the anteriorly placed pin (Figure 1C). The incisions should just pass through the cuticle. 
		NOTE: The three incisions from steps 1.1.7 and 1.1.8 combined should resemble an &#34;<img alt="Equation 1" src="/files/ftp_upload/23255/23255eq01.jpg" style="margin: auto;" />&#34; when completed,&#160;i.e., a left- and right-hand flap on the dorsal side of the larval body should be produced.</li>
		<li>Carefully clean out the internal organs with the forceps. Adding a few forceful drops of PBS will help displace the organs out of the larval body, thus making it easier to remove them. Avoid poking the larval body as it will cause damage to the body wall muscles.</li>
		<li>Unfurl the larval body open and pin the corners down (Figure 1D). When pinning, stretch the body wall both horizontally and vertically to form an evenly tensioned rectangle (see&#160;Figure 1G&#160;for the shape), taking care not to tear the body wall muscles in the process.</li>
		<li>Finish removing any remaining internal organs (Figure 1E).</li>
	</ol>
	</li>
	<li>Fixation and Permeabilization
	<ol>
		<li>Immerse the pinned body walls in several drops of Bouin&#39;s Solution. Incubate for 15 min on ice. Alternatively, use 4% paraformaldehyde (PFA) as an alternative fixative; incubate for 30 min.</li>
		<li>Rinse three times with Phosphate Buffer Saline with Triton (PBT).</li>
		<li>Using fine forceps, carefully remove the pins and transfer the body walls by their corners into a siliconized 0.65 ml microcentrifuge tube.</li>
		<li>Store the body walls in PBT at 4 &#176;C until ready for PLA. For optimal results, start immunostaining the body walls within a day or two of dissection. 
		NOTE: To save on reagents and to ensure that all body walls are treated equally during the assay, different genotypes can be placed into a single tube. Genotypes can be distinguished by cutting the corners of the body walls differently (see&#160;Figure 1F&#160;for example).</li>
	</ol>
	</li>
</ol>
2. Immunohistochemistry
NOTE: Immunostaining of third instar larval body walls was performed as previously described but with some modifications. 
NOTE: Perform all steps at room temperature and with gentle agitation unless otherwise stated.
<ol>
	<li>Blocking
	<ol>
		<li>Wash the body walls with PBT three times for 10 min each.</li>
		<li>Block with 1% Bovine Serum Albumin (BSA) for 1 hr.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Incubate the body walls with mouse and rabbit primary antibodies against the two proteins of interest (diluted in 1% BSA) for 2 hr at room temperature, or overnight at 4 &#176;C. In this case, use 1:10 mouse anti-Dlg (Dlg = discs large) and 1:250 rabbit anti-HtsM (HtsM: Hu-li tai shao mutant). Antibodies against markers that are not made in mouse or rabbit can also be included &#8212;&#160;e.g., use 1:200 goat anti-Hrp (Hrp = horseradish peroxidase) to delineate the neuronal membranes.</li>
		<li>Wash with PBT three times for 10 min each.</li>
		<li>Incubate with fluorophore-conjugated secondary antibodies to detect the markers (diluted in 1% BSA) for 2 hr at room temperature, or overnight at 4 &#176;C. As the PLA signal is later visualized with a red fluorophore, another fluorophore must be used to detect the marker &#8212;&#160;e.g., use 1:200 Fluorescein isothiocyanate (FITC)-conjugated anti-goat to detect the goat anti-Hrp antibody. Due to the use of light-sensitive reagents, keep the tubes in the dark from this point onwards. 
		NOTE: The kit that is used allows PLA to be performed between primary antibodies raised in mouse and rabbit, with the signal visualized on the red channel under confocal microscopy. If desired, other kits are available that allow the assay to be done with primary antibodies raised in other species, and the signal visualized on other channels.</li>
	</ol>
	</li>
</ol>
3. Proximity Ligation Assay
NOTE: Perform all steps at room temperature and with gentle agitation unless otherwise stated.
<ol>
	<li>PLA Probes
	<ol>
		<li>Wash the body walls with PBT three times for 10 min each.</li>
		<li>Incubate with PLA probes (1:5 dilution each in 1% BSA) for 2 hr at 37 &#176;C. In this case, use 40 &#181;l of PLA probe anti-mouse MINUS, 40 &#181;l of PLA probe anti-rabbit PLUS and 120 &#181;l of 1% BSA to ensure the proper immersion and mixing of 5-10 body walls. Up to a 1:25 dilution of the PLA probes can still result in an adequate signal-to-noise ratio (i.e., for this experiment).</li>
	</ol>
	</li>
	<li>Ligation
	<ol>
		<li>Wash the body walls with Wash Buffer A twice for 5 min each.</li>
		<li>Incubate with Ligation solution (1:40 dilution of Ligase in Ligation buffer) for 1 hr at 37 &#176;C. In this case, use 5 &#181;l of Ligase, 40 &#181;l of 5 Ligation buffer and 155 &#181;l of high purity water to ensure the proper immersion and mixing of 5-10 body walls.</li>
	</ol>
	</li>
	<li>Amplification
	<ol>
		<li>Wash the body walls with Wash Buffer A twice for 2 min each.</li>
		<li>Incubate with Amplification solution (1:80 dilution of Polymerase in Amplification buffer) for 2 hr at 37 &#176;C. In this case, use 2.5 &#181;l of Polymerase, 40 &#181;l of 5 Amplification buffer and 157.5 &#181;l of high purity water to ensure the proper immersion and mixing of 5-10 body walls.</li>
	</ol>
	</li>
	<li>Preparation for Imaging
	<ol>
		<li>Wash the body walls with Wash Buffer B twice for 10 min each.</li>
		<li>Wash with 0.01x Wash Buffer B once for 1 min.</li>
		<li>Equilibrate in a few drops of mounting solution for at least 30 min before mounting, or store overnight at 4 &#176;C.</li>
		<li>Using fine forceps, carefully transfer the body walls onto a platform slide with their cuticles facing down. Position the body walls in rows and in the same orientation within a drop or two of mountant. Place a 22 mm x 40 mm coverslip over the preparation taking care not to generate air bubbles, then seal the slide with clear nail polish.</li>
		<li>Store the slides in the dark at -20 &#176;C until ready for confocal imaging.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Forceps (fine #5)</td>
			<td>Almedic</td>
			<td>A10-704</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard Disc</td>
			<td>World Precision Instruments</td>
			<td>SYLG184</td>
			<td>Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (e.g. use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60x15mm petri dish lid when dissecting.</td>
		</tr>
		<tr>
			<td>Minutien Pins (0.0125 mm tip diameter)</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissection Scissors (ultra fine)</td>
			<td>Fine Science Tools</td>
			<td>15200-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Platform Slides</td>
			<td></td>
			<td></td>
			<td>Glue two 22x22mm coverslips onto a microscope slide with clear nail polish, leaving a &#60;20mm gap in between for sample mounting.</td>
		</tr>
		<tr>
			<td>w1118</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>3605</td>
			<td></td>
		</tr>
		<tr>
			<td>hts01103</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>10989</td>
			<td>Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal.</td>
		</tr>
		<tr>
			<td>1x PBS (Phosphate Buffered Saline): 3mM NaH2PO4, 7mM Na2HPO4, 130mM NaCl, pH 7.0</td>
			<td></td>
			<td></td>
			<td>NaH2PO4&#160;(Caledon Laboratories - 8180-1), Na2HPO4&#160;(Caledon - 8120-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr>
			<td>Bouin&#39;s Solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT10132</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA (Paraformaldehyde): 4% PFA in 1x PBS</td>
			<td></td>
			<td></td>
			<td>PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.&#160;</td>
		</tr>
		<tr>
			<td>1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton</td>
			<td></td>
			<td></td>
			<td>Triton X-100 (Sigma-Aldrich - T8787)</td>
		</tr>
		<tr>
			<td>1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT</td>
			<td></td>
			<td></td>
			<td>BSA (Bioshop Canada - ALB001). Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>mouse anti-Dlg (Discs large)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td>Use at a 1:10 dilution in 1% BSA.</td>
		</tr>
		<tr>
			<td>rabbit anti-HtsM (Hu-li tai shao)</td>
			<td></td>
			<td></td>
			<td>Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.</td>
		</tr>
		<tr>
			<td>rabbit anti-Pak (p21-activated kinase)</td>
			<td></td>
			<td></td>
			<td>Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.</td>
		</tr>
		<tr>
			<td>mouse anti-Wg (Wingless)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4D4</td>
			<td>Use at a 1:5 dilution in 1% BSA.</td>
		</tr>
		<tr>
			<td>goat anti-Hrp (Horseradish peroxidase)</td>
			<td>Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td>123-065-021</td>
			<td>Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr>
			<td>FITC-conjugated donkey anti-goat</td>
			<td>Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td>705-095-003</td>
			<td>Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr>
			<td>Duolink In Situ PLA Probe anti-mouse MINUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92004</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ PLA Probe anti-rabbit PLUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92002</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink In Situ Detection Reagents Red</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92008</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Wash Buffer A: 0.01M Tris, 0.15M NaCl, 0.05% Tween 20, pH 7.4</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82049</td>
			<td>Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)</td>
		</tr>
		<tr>
			<td>1x Wash Buffer B: 0.2M Tris, 0.1M NaCl, pH 7.5</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82049</td>
			<td>Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr>
			<td>0.01x Wash Buffer B: 2mM Tris, 1mM NaCl, pH 7.5</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82049</td>
			<td>Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr>
			<td>Duolink In Situ Mounting Medium with DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82040</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of protein-protein interactions in drosophila larvae using a proximity ligation assay

Source: Wang, S., et al. <a href="https://app.jove.com/v/52139">Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay.</a> J. Vis. Exp. (2015).
This video demonstrates the visualization of protein-protein interactions at the Drosophila neuromuscular junction (NMJ) using Proximity Ligation Assay (PLA). After isolating the body walls, interacting proteins are probed with primary antibodies and PLA-conjugated secondary antibodies. The proximity of the interacting proteins facilitates the ligation of probes with connector oligonucleotides, forming circular DNA, which is then amplified by rolling circle amplification. Fluorescent probes bind to the amplified DNA, allowing the visualization of protein-protein interactions at the NMJ.

<img alt="Figure 1" src="/files/ftp_upload/23255/23255_Figure1.jpg" /> 
Figure 1: Preparation of third instar larval body walls. (A-F)&#160;Schematic drawings representing a body wall dissection.&#160;(A)&#160;Place a single larva onto a sylgard disc with its dorsal side facing up so that the two tracheal tracts are visible.&#160;(B)&#160;Pin the larva down at the anterior and posterior ends with minutien pins, stretching the larva o...

Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay

Source: Wang, S., et al. Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay. J. ...

Take the body wall of Drosophila larvae, consisting of muscles innervated by motor neurons to form neuromuscular junctions or NMJs.
The postsynaptic muscle membrane at the NMJ contains interacting membrane-bound proteins, which are labeled with primary antibodies.
Add proximity ligation assay, or PLA, probes containing secondary antibodies conjugated to single-stranded oligonucleotides. Incubate for the probes to bind to the primary antibodies.
Wash away any unbound probes.
Incubate with ligation buffer containing connector oligonucleotides and ligase enzymes.
The proximity of the interacting proteins facilitates connector binding to probes. Ligase then seals the gaps, creating circular DNA.
Wash away any unbound connectors.
Incubate with an amplification solution containing polymerase, which amplifies the circular DNA through rolling circle amplification.
Fluorophore-tagged detector probes in the solution anneal to the amplified DNA, and upon excitation produce a fluorescent signal.
Mount the body wall on a slide to visualize fluorescently labeled protein-protein interactions at the NMJ.

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Neuromuscular Disorders

27 Views. Source: Wang, S., et al. Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay. J. Vis. Exp. (2015). This video demonstrates the visualization of protein-protein interactions at the Drosophila neuromuscular junction (NMJ) using Proximity Ligation Assay (PLA). After isolating the body walls, interacting proteins are probed with primary antibodies and PLA-conjugated secondary antibodies. The proximity of the interacting prote...

Video: Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay - Concept

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

JoVE Journal

Biochemistry

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay

Transcript

Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Visualization and Quantification of TGFβ/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay