Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Podsumowanie

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Streszczenie

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

Wprowadzenie

The mammary gland is an atypical mammalian exocrine organ whose main function is to produce milk to feed newborns. The development of the mammary tissue occurs mainly after birth and is characterized by a unique process in which the epithelium invades the surrounding stroma. This tissue undergoes many changes (growth, differentiation and regression), especially during the adult life, concomitantly with variations in reproductive status (Figure 1). In addition to the overall morphology of the tissue, the proportions of different cell types as well as their arrangement within the mammary gland dramatically change during development^1-5.

During embryonic life, the mammary epithelium derives from mammary milk lines, which is defined by a slight thickening and stratification of the ectoderm, between the fore and hind limbs on each side of the midline around embryonic day 10.5 (E10.5) (Figure 1A). On E11.5, the milk line breaks up into individual placodes, which are symmetrically positioned along the mammary milk line at reproducible locations, and the surrounding mesenchyme starts to condense. The placodes begin to sink deeper into the dermis and the mammary mesenchyme organizes in concentric layers around the mammary bud (E12.5-E14.5). As of E15.5, the mammary epithelium, starts to proliferate and elongate to form the primary sprout that pushes through the mammary mesenchyme towards the fat pad. The primary sprout develops a hollow lumen with an opening to the skin, marked by the formation of the nipple sheath. On E18.5, the elongating duct has grown into the fat pad and has branched into a small arborized ductal system encompassed in the fat pad. Development is essentially arrested and the rudimentary mammary gland remains morphogenetically quiescent until puberty. In the male embryo, the activation of androgen receptors leads to the degeneration of the buds, which disappear by E15.5. As of E18, mammary development ceases until puberty^6-9.

At birth, the mammary gland harbors a rudimentary ductal system that elongates and branches slowly (isometric growth). At the onset of puberty, spherical structures located at the tips of the ducts called the terminal end buds (TEBs), are formed of an outer layer of cap cells and a multilayered inner core of cells (body cells). These structures are highly proliferative and infiltrate the surrounding stromal tissue in response to hormonal cues. Proliferation within the TEBs results in ductal elongation, coupled with branching morphogenesis. This process leads to the establishment of a basic epithelial arborized network emanating from the nipple (Figure 1B, puberty). At ~10-12 weeks after birth, when the epithelium has invaded the whole fat pad, its expansion stops and the TEBs disappear. Ductal development then undergoes dynamic changes, i.e., successive proliferation and regression of epithelial cells according to estrous cycles¹⁰ (Figure 1B, adult).

From the onset of gestation, the mammary tissue undergoes important growth and morphological changes to prepare for lactation. The mammary epithelium extensively proliferate and differentiate, leading to a highly branched tubulo-alveolar network. Concomitantly, mammary epithelial cells (MECs) become polarized and able to synthesize and secrete milk products. MECs organize into numerous alveolar structures (acini) that are surrounded by contractile myoepithelial cells and incorporated in a stroma composed of connective and adipose tissues, blood vessels and nerve terminals (Figure 1B, pregnancy). Furthermore, the basal side of MECs is in close contact with the basement membrane (extracellular matrix), and interactions between these two entities tightly regulate both morphogenesis and secretory function of the mammary epithelium^11-13. 

All these processes rely on the action of various environmental cues, of which the most important are hormones14, paracrine factors and the extracellular matrix. For example, progesterone induces extensive side-branching¹⁵ and alveologenesis that, in combination with prolactin (PRL)^16,17, promotes and maintains the differentiation of the alveoli. In addition to steroids and PRL18, cytokines and signaling pathways associated with development (Wnt and Notch signaling pathways) are also involved in mammary lineage commitment and development^19-21. At the end of pregnancy, the luminal MECs begin to produce a protein-rich milk known as colostrum in the lumen of the alveoli. In addition, progesterone acts on the epithelial permeability and since the tight junctions are still open, colostrum is also found in the maternal blood stream. 

After parturition, the mammary epithelium takes up almost all of the mammary gland volume and is highly organized (Figure 2, mammary epithelium). Milk-producing units, namely alveoli (Figure 2, alveolus), are formed by a monolayer of polarized mammary epithelial secretory cells (MESCs), with their apical plasma membrane delimiting the lumen. Alveoli arrange themselves into lobules that are grouped into lobes connected to ducts that drain milk to the outside milieu (Figure 2, lobe). Lactation occurs, i.e., MESCs start to secrete abundant amounts of milk, primarily triggered by the drop in placental hormones (mainly progesterone) (Figure 1B, lactation). Milk protein genes are activated in a defined temporal time course ranging from pregnancy to lactation^9,22,23, chiefly in response to pituitary PRL released at the time of suckling. Concomitantly, contacts between MESCs and the extracellular matrix both stimulate milk protein synthesis through signals that are mediated via the interactions between cellular integrins and laminin^24,25, and suppress apoptosis in MESCs^26,27. These signaling pathways result in the activation of milk protein gene promoters²⁸ through the activation of specific transcription factors²⁹. Cell-cell contacts are also important for some aspects of differentiation including the establishment of apical polarity and the vectorial secretion of milk products. Tight junctions rapidly close after the beginning of lactation and MESCs finely orchestrate the uptake of molecules from the blood as well as the synthesis, transport and secretion of milk components, in response to the nutritional requirements of neonates. At the time of suckling, the contraction of the myoepithelial cells surrounding the alveoli occurs in response to oxytocin and leads to milk ejection through the ducts and into the nipple. Milk is a complex fluid that contains proteins (mostly caseins), sugars (mainly lactose), lipids and minerals, as well as bioactive molecules such as immunoglobulins A (IgA), growth factors and hormones. Caseins are synthesized, assembled in supramolecular structures, namely casein micelles, transported along the secretory pathway, and then released by exocytosis, i.e., the fusion of casein-containing secretory vesicles (SVs) with the apical plasma membrane of MESC (Figure 2). 

Intracellular traffic relies on material exchanges between membranous compartments and involves Soluble N-ethylmaleimide-Sensitive Fusion (NSF) Attachment Protein (SNAP) Receptor (SNARE)^30,31. The SNARE proteins family is subdivided in vesicular SNAREs (v-SNAREs), present in the vesicle membrane, and target SNAREs (t-SNAREs), localized on the target membranes. By zipping through their coiled-coil domains, v- and t-SNAREs assemble to form a highly stable four-helix bundle complex, referred to as the SNARE complex. This complex promotes the fusion of two opposing lipid bilayers by gradually bringing them into close proximity^30,32. Afterwards, SNARE complexes are dissociated by the NSF adenosine triphosphatase and its adaptor protein SNAP and SNARE proteins are recycled back to their compartment of origin³³. Interestingly, each SNARE protein predominantly resides in distinct cellular compartments and SNARE pairing may contribute to the specificity of intracellular fusion events³⁴. Previous studies suggest that at least Synaptosomal-Associated Protein 23 (SNAP23) and Vesicle-Associated Membrane Protein 8 (VAMP8), and syntaxins (Stx) -7 and -12 play a role in casein exocytosis ^35,36. These proteins have also been found in association with the lipid fraction of milk, i.e., milk fat globules (MFGs)³⁷. The current prevailing model postulates that cytoplasmic lipid droplets (CLDs) are formed by the accumulation of neutral lipids (mainly triacylglycerols and sterol esters) and cholesterol derived from the maternal diet between the two leaflets of the endoplasmic reticulum (ER) membrane^38-41. Large CLDs are formed, at least in part, by the fusion of smaller CLDs while being transported to the apical side of MESCs where they are released as MFGs (1-10 µm in diameter) by budding, being enwrapped by the MESC apical plasma membrane^40-42. Lactation ceases after pups are weaned and the MESCs progressively die by apoptosis, leading to the regression of the mammary tissue back to a pubertal state (Figure 1B, involution).

Immunofluorescence (IF) is a common analytical laboratory method used in almost all aspects of biology, both in research and in clinical diagnostics. IF techniques can be performed on tissue sections (immunohistochemistry, IHC) or cell (immunocytochemistry, ICC) samples. This powerful approach relies on the use of fluorescent-labeled antibodies that specifically bind (directly or indirectly) to the antigen of interest, thus allowing the visualization of its tissue distribution through fluorescence microscopy. Fluorescence signals mostly depend on the quality and concentration of the antibodies and proper handling of the specimen. A simple indirect immunofluorescence (IIF) protocol is presented to detect milk products (caseins and MFGs) and proteins involved in milk product secretion (butyrophilin (BTN1), SNARE proteins) on frozen sections of mouse mammary tissue (Figure 3). While this protocol provides a complete IHC overview, ranging from tissue collection to image post-treatment, critical and optional steps as well as some technical recommendations are also presented and discussed.

Protokół

CD1 mice were bred at INRA (UE0907 IERP, Jouy-en-Josas, France). All ethical aspects of animal care complied with the relevant guidelines and licensing requirements laid down by the French Ministry of Agriculture. The procedures used were approved by the local ethics committee (agreement 12/097 from the Comethea Jouy-en-Josas/AgroParisTech).

1. Mammary Gland Sample Preparation

1. Mouse mammary gland dissection
   1. Euthanize mice at day 10 of lactation by cervical dislocation and pin the animal down with its abdomen facing up. 
   2. Wet the ventral area with ethanol and dry it with a paper towel. 
   3. Using forceps, pull up the abdominal skin between the two hind legs and make an incision (through the skin only) of about 1 cm with sharp scissors. Starting from this first incision, then use scissors to cut the skin up to the neck on the mouse. Pull the skin away from the peritoneum and pin down one side of the skin at a time, stretching it taught. 
   4. Collect the abdominal and the inguinal mammary glands by pushing them away from the skin with a swab and finally pulling or cutting them away from the peritoneum. 
      Note: At this step Carmine staining can be performed in order to visualize the mammary epithelium within the entire gland⁴³. This approach can be useful to analyze the global morphology of the mammary gland under various conditions (physiological developmental stages, diseases, in vivo treatments).
   5. Remove the lymph node located at the junction of the abdominal and the inguinal glands⁴⁴.
2. Mammary tissue fixation
   1. Cut the mammary tissue into 3 mm³ fragments with a scalpel and immediately rinse these fragments in a phosphate buffered saline (PBS) solution, pH 7.4, in order to remove as much milk as possible. 
   2. Quickly dry the fragments on a paper towel and put them in a cold PBS solution containing 4% paraformaldehyde (PFA, HCHO, 32% formaldehyde solution, CAUTION) for 10 to 15 min on ice.
      Note: This is enough time to allow subsequent analysis on mammary tissue slices by IIF36 and/or in situ hybridization⁴⁵. However, as aldehyde fixatives penetrate rather slowly in tissue pieces (~1-3 mm per hour), this time may be extended to ensure an optimal fixation of the tissue sample. Alternatively, fix tissues in vivo by perfusing an anesthetized animal with a fixative solution (not detailed in the present study).
3. Sucrose infusion
   1. Quickly rinse the mammary fragments in cold PBS and immerse them in a cold PBS solution containing 40% sucrose (D-saccharose, C12H22O11, Mr 342.3 g/mol) for 16 to 48 hr at 4 °C under gentle shaking.
4. Tissue embedding
   Note: At this step, mammary fragments can be re-cut in order to make smaller fragments (2-3 mm³) or to adjust their shape. 
   1. Properly label the plastic molds and fill a third of the volume of the mold with OCT compound, maintained at RT. Place one fragment (2-3 mm³) of mammary tissue per mold and cover it with OCT compound. 
   2. Place the molds at the surface of the liquid nitrogen (on a sheet of aluminum or using a metallic sieve) and allow the product to freeze.
      Note: It must become solid and white before immersing the mold in liquid nitrogen.
5. Store the frozen samples at -80 °C until tissue sections are performed.

2. Frozen tissue Sectioning

Note: A cryostat, which is essentially a microtome inside a freezer, is required to make frozen tissue sections. A lower temperature is often required for fat or lipid-rich tissues such as virgin mammary gland.

1. Adjust the temperature of the cryostat to -26 °C and wait until it has stabilized. Maintain the frozen tissue block at -26 °C throughout the entire sectioning procedure. Absolutely avoid thawing the tissue at any time during the procedure.
2. Cool the razor blade, the cutting support, the anti-roll device and the brush to -26 °C by placing them in the cryostat for at least 10 min. Also place a slide box inside the cryostat in order to be able to store glass slides as the sections are made. 
3. Properly label the glass slides that will be used to collect the tissue sections and maintain them at RT; otherwise tissue sections will not adhere to them. Remove the sample from the mold inside the cryostat. 
   Note: Using positively charged glass slides will greatly favor the adhesion of fresh frozen tissue sections due to higher electrostatic attraction.
4. Cover the surface of a metal tissue disc with OCT compound (maintained at RT) and push the frozen sample onto it. Place the wet mount inside the cryostat and let it cool for at least 15 min.
5. Place the wet mount in the disc holder of the cryostat. Adjust the cut thickness to 5-6 µm and, if possible, use a new sharp blade or at least change the area on the blade used to cut each sample since some tissues will quickly dull it. 
6. Adjust the position of the anti-roll device over the razor blade by making cuts of the mounting medium until the slices are formed evenly and correctly. Ideally, the anti-roll device will step over the razor blade by about 1 mm. 
7. Once the settings are correct, perform tissue sections by turning the wheel in a continuous uniform motion. Unless the temperature is ideal, a tissue section will, by nature, try to curl up. 
   1. Use a brush to grab and maneuver the section across the stage in order to place it as desired on the glass slide. Use the brush to clean up the remains possibly present on the frozen tissue block and/or the razor blade. 
8. Pull the tissue section toward the user and avoid pressing it onto the cryostat stage. Avoid pressing the tissue section onto the cryostat stage as it may lead to the adhesion of the tissue slice on the stage and thus the inability to recover it with the glass slide.
9. Retrieve tissue sections one by one by picking them up at the surface of a glass slide, by holding it above the section and angling it down to touch the tissue section.
   Note: Tissue sections quickly adhere to the warm glass due to static attraction. If several tissue sections are placed on the same slide, be careful not to overlap them and to space them enough to be able to individually enclose them in a hydrophobic circle (see section 3.1.1.).

3. Indirect Immunofluorescence

1. Locating sections
   1. Use a hydrophobic barrier pen to draw a hydrophobic circle around slide-mounted tissue. Let the circle dry for approximately 1 min at RT. Draw a line around the tissue sections with a fine black permanent marker as well, but on the side of the glass slide opposite to the one where the tissue sections are. 
      Note: This circle is water-repellent and acetone- and alcohol-insoluble. It therefore provides a barrier to aqueous solutions used during the IHC procedure and reduces the volume of required reagents. 
   2. Rehydrate tissue sections by covering them with a drop of ~250 µl of PBS for a few minutes at RT. Fix tissue sections by covering them with ~250 µl of a freshly prepared 3% PFA solution in PBS for 10 to 15 min. 
      Note: Optionally in this case, use an aldehyde quenching solution (50 mM ammonium chloride (NH₄Cl, Mr 53.5 g/mol) in PBS or 0.1M glycine (C₂H₅NO₂, Mr 75.07 g/mol) in PBS) to stop the fixation reaction. Simple and abundant PBS washing is generally sufficient to remove unreacted aldehyde.
2. Antigen retrieval (optional)
   Note: While most antigens can be detected without antigen retrieval (AR), others will be observed only if this step is performed. In some cases, AR also allows the enhancement of the observed signal.
   1. Place the AR solution (100 mM Tris (C₄H₁₁NO₃, Mr 121.14) 5% urea (NH₂CONH₂, Mr 60.06) pH 9.6) in a beaker. The volume of AR solution must be sufficient to completely cover the glass slides placed in a glass holder. 
   2. Preheat the AR solution to 95 °C by monitoring the temperature with a thermometer and then place the glass slides on a suitable rack, immerge the rack in the hot buffer, cover to limit evaporation and incubate for 10 min at 95 °C. 
   3. Remove the beaker from the water bath and leave the glass slides for another 10 min in the buffer.
3. Immunodetection
   1. Rinse tissue sections with PBS (~250 µl/section) and saturate them with a solution of 3% bovine serum albumin (BSA, ~250 µl/section) in PBS for at least 30 min at RT. 
   2. Put 30-50 µl of the primary antibody diluted in PBS containing 2% BSA on each tissue section.
      Note: This volume is enough to form a drop that completely covers the tissue section.
   3. Place the same volume of the diluent (2% BSA in PBS) alone on a tissue section to perform a negative control without primary antibody.
      1. Systematically include this negative control in each IHC experiment and perform for each secondary antibody used to estimate the background of the experiment (non-specific labeling due to the secondary antibody and/or the tissue auto-fluorescence). Other types of positive or negative controls can also be performed to ensure the specificity of the labeling (see discussion).
   4. Place the glass slides in a humidified box O/N at 4 °C.
      Note: Primary antibodies used were mouse monoclonal anti-cytokeratin 8 (CK8, 1:50 dilution), mouse monoclonal anti-cytokeratin 14 (CK14, 1:50 dilution), rabbit polyclonal anti-mouse casein (#7781, 1:50 dilution, generously provided by M.C. Neville, University of Colorado Health Sciences Center, CO, USA), rabbit polyclonal anti-BTN1 (1:300 dilution, generously provided by I.H. Mather, Department of Animal and Avian Sciences, University of Maryland, College Park, MD, USA), rabbit polyclonal anti-Stx6 (1:50 dilution, generously provided by S. Tooze, Cancer Research UK, London Research Institute, London, UK) and rabbit polyclonal anti-VAMP4 (1:50 dilution). 
   5. Thoroughly wash tissue sections with PBS at least four times for 10 min at RT.
   6. Dilute the appropriate secondary antibody (rhodamine-conjugated goat anti-rabbit IgG (H + L), 1:300 dilution) in PBS containing 2% BSA, place 30-50 µl of this solution on all tissue sections, and incubate for 1.5 hr at RT.
      1. Since fluorochromes are light-sensitive molecules, do not expose tissue sections to light until their analysis. For IIF on tissue sections, favor secondary antibodies coupled to a red fluorophore since cell membranes tend to generate a green auto-fluorescence that can interfere with low labeling. Moreover, choosing a red fluorophore-coupled secondary antibody allows the concomitant labeling of neutral lipids (see below). 
   7. Thoroughly wash the tissue sections with PBS at least four times for 10 min at RT. 
4. Post-fixation (optional)
   1. For some experiments, perform post-fixation by incubating the samples with 2% PFA diluted in PBS for 10 min at RT in order to stabilize the antigen/antibody scaffolds. However, this step can be dispensed with in most cases.
5. Neutral lipids and DNA counterstaining
   1. To visualize CLDs and MFGs, color neutral lipids by incubating tissue sections in 30-50 µl of a PBS solution containing 3 µg/ml of bodipy 493/503 for 10 min at RT. Rapidly rinse tissue sections twice with PBS.
   2. Counterstain nuclear DNA with 30-50 µl of a PBS solution containing 3 µM of DAPI (4-6-diamidino-2-phenylindole, 5 mg/ml stock solution) for 10 min at RT. Wash the tissue sections twice with PBS before mounting the slides for observation.
6. Section mounting
   1. Remove PBS and place a drop of mounting medium on each tissue section. 
   2. Place one side of the cover slip at an angle against the slide, making contact with the outer edge of the liquid drop and then lower the cover slowly, avoid air bubbles. Allow the liquid to spread between the glass slide and the cover slip for a few minutes and then remove the excess of mounting medium with a paper towel.
   3. Seal the cover slip to the glass slide with nail polish and store tissue sections at 4 °C to prevent their exposure to light until observation.

4. Fluorescence Observation and Image Acquisition

Note: A fluorescence microscope equipped with a camera controlled by image acquisition software is required to observe the IHC results.

1. Before acquiring images, check the intensity of the labeling and evaluate the background of the experiment by looking at the negative controls. Acquire pictures of each fluorescent label (color channel) individually.
2. Acquire all pictures, including those of the corresponding controls, in the same conditions (exposure and general settings) for each color channel.
3. Conventional microscopy
   1. Perform epifluorescence microscopy with a microscope equipped with standard filters for fluorescein isothiocyanate (FITC, green), rhodamine (red) and DAPI (blue) emissions, ×20 to ×63 (oil-immersion, NA 1.3) objectives and a DP50 imaging camera.
4. Confocal microscopy
5. Perform confocal microscopy with a microscope equipped with the ZEN software, using ×20 to ×63 (oil-immersion, NA 1.4) objectives and the 488- and 568-nm excitation wavelengths of the laser.

5. Image Treatment

Note: All image post-treatments are performed using the ImageJ free software (http://imagej.nih.gov/ij/).

1. Superimpose image (merge)
   1. Open the images acquired in each channel that will be combined (File/Open). If working with 8-bit grayscale images, attribute artificial color to each channel using the lookup table (Image/Lookup Tables). 
   2. Generate the composite picture from grayscale or colored images by using the command “Merge channels” (Image/Color/Merge Channels) and then attributing a color to each channel. 
   3. Perform image stacks superimposition in the same way by opening stacks acquired in each channel that will be combined (File/Open) and using the command “Merge channels” (Image/Color/Merge Channels) to attribute a color to each channel. Save the composite stack as an image sequence or as a movie (see section 5.4).
2. Image stack Z projection
   1. Use the Z projection function (Image/Stack/Zproject, Max Intensity) to provide a two-dimensional view of all the pictures of an image stack by projecting them along the axis perpendicular to the image plane (z-axis). The “Maximum Intensity” option creates an image in which each pixel contains the maximum value over all images in the stack. This generates a single image allowing the visualization of all the staining observed through the whole image stack for a particular channel or after the superimposition of several channels.
3. Image stack 3D projection
   1. Use the 3D projection command (Image/Stack/3D project, Brightest Point, y-axis) to generate a sequence of projections of a rotating volume onto a plane. The visual rendering of surfaces and internal structures depends on both the projection method (nearest-point, brightest point (used here), or mean-value) and the visualization parameters selected. Each frame of the animated sequence is the result of projecting from a different viewing angle. 
   2. Rotate the created 3D image around each of the three orthogonal axes (the y-axis was selected here). Save the sequence produced as a single image or a movie.
4. Image stack to movie conversion
   1. Open an image stack (File/Open) and save it as a movie in .AVI format by using the command “AVI” (File/Save As/AVI).

Wyniki

The mammary gland is a subcutaneous gland located along the ventral structure of both the thorax and the abdomen in rodents. The location of the five pairs of glands of the mouse during gestation is shown in Figure 4. The morphology of the mammary gland dramatically changes during its development, reflecting functional modifications required to prepare for full lactation (Figure 1B). In virgin or nulliparous animals, the mammary gland consists of a sparsely branched ductal e...

Dyskusje

IHC is a relatively simple and straightforward experimental method to localize antigen in tissue sections, which depends primarily on specific epitope-antibody interactions. Although a large number of protocols are used to localize a protein by IIF, the core of these procedures is almost always the same. However, there are some critical aspects that can strongly influence the result and must therefore be optimized for each individual IHC study. The most challenging aspect of this approach is to determine the best experim...

Materiały

Name	Company	Catalog Number	Comments
Dissection	Company	Catalog Number	Comments/Description
Pins
Ethanol
Scissors
Scalpel and adapted blades
Ice
Towel paper
Tissue sample preparation	Company	Catalog Number	Comments/Description
Phosphate Buffered Saline (pH7.4)	Sigma	P-3813
Paraformaldehyde (PFA, 32% EM grade, 100 ml)	Electron Microscopy Sciences	15714-S	personnal protection equipment required WARNING: this product will expose you to Formaldehyde Gas, a chemical known to cause cancer
OCT compound/Tissue Tek	Sakura	4583
Sucrose (D-saccharose)	VWR	27480.294
Plastic molds	Dominique Dutscher	39910
Liquid nitrogen
Cryostat/sample support	Leica	CM3050S
Razor blades (SEC35)	Thermo Scientific	152200
Slide box
Glass slides Superfrost/Superfrost Ultra Plus	Thermo Scientific	10143560W90/1014356190
Brushes
IHC	Company	Catalog Number	Comments/Description
Super Pap Pen	Sigma	Z377821-1EA
Permanent marker (black)
50 mM NH4Cl in PBS	Sigma	A-0171
0.1 M glycine in PBS	VWR	24403.367
Antigen Retrieval solution: Tris 100 mM 5% urea pH9.6
Heater (up to 100°C)
Bovine Serum Albumin (BSA)	Sigma	A7906-100G
Vectashield (anti-fading mounting medium) without DAPI/with DAPI	Vector Laboratories	H-1000/H-1200
Glass coverslips 22x50mm (microscopy grade)	VWR	CORN2980-225
Nail polish
Primary antibodies	Company	Catalog Number	Comments/Description
Rabbit anti-mouse caseins (#7781; 1:50 dilution)	generously gifted by M.C. Neville (University of Colorado Health Sciences Center, USA)
Mouse anti-cytokeratin 8 (CK8, clone 1E8, 1:50 dilution)	Biolegend (Covance)	MMS-162P
Mouse anti-cytokeratin 14 (CK14, cloneLL002, 1:50 dilution)	Thermo Scientific	MS-115-P0/P1
Rabbit anti-butyrophilin (1:300 dilution)	generously gifted by I.H. Mather (Department of Animal and Avian Sciences University of Maryland College Park, USA)
Rabbit anti-Stx6 (1:50 dilution)	generously gifted S. Tooze (Cancer Research UK, London Research Institute, London, UK)
Rabbit anti-VAMP4 (1:50 dilution)	Abcam	ab3348
Secondary antibodies	Company	Catalog Number	Comments/Description
Rhodamine-conjugated goat anti-rabbit IgG (H + L) (1:300 dilution)	Jackson ImmunoResearch Laboratories	111-025-003
Counterstains	Company	Catalog Number	Comments/Description
Bodipy 493/503	Life Technologies (Molecular Probes)	D-3922
DAPI (4-6-diamidino-2-phenylindole)	Life Technologies (Molecular Probes)	D-1306
Observation/Image capture	Company	Catalog Number	Comments/Description
conventional fluorescence microscope	Leica Leitz DMRB microscope		Standard filters for FITC, Rhodamine and DAPI emissions,                     ×63 oil-immersion objective (NA 1.3), DP50 imaging camera (Olympus), CellˆF software (Olympus)
Laser Scanning Microscope (confocal microscopy)	Zeiss LSM 510 microscope		Plan-Apochromat ×63 oil-immersion objective (NA 1.4),                  CLSM 510 software,               Confocal facilities, MIMA2 Platform, INRA Jouy-en-Josas, France, http://mima2.jouy.inra.fr/mima2)
Image treatment	Company	Catalog Number	Comments/Description
ImageJ 1.49k software			Free software