The authors are grateful to the INRA MIMA2 imaging core facility (INRA, UMR1198, Jouy-en-Josas) and to the staff of the IERP unit (UE 0907, INRA, Jouy-en-Josas) for animal care and facilities. We would also like to thank I.H. Mather, M.C. Neville and S. Tooze for providing us with very useful antibodie.

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
<ol>
	<li>Mouse mammary gland dissection
	<ol>
		<li>Euthanize mice at day 10 of lactation by cervical dislocation and pin the animal down with its abdomen facing up.&#160;</li>
		<li>Wet the ventral area with ethanol and dry it with a paper towel.&#160;</li>
		<li>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.&#160;</li>
		<li>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.&#160; 
		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).</li>
		<li>Remove the lymph node located at the junction of the abdominal and the inguinal glands44.</li>
	</ol>
	</li>
	<li>Mammary tissue fixation
	<ol>
		<li>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.&#160;</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Sucrose infusion
	<ol>
		<li>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 &#176;C under gentle shaking.</li>
	</ol>
	</li>
	<li>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.&#160;
	<ol>
		<li>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.&#160;</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Store the frozen samples at -80 &#176;C until tissue sections are performed.</li>
</ol>
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.
<ol>
	<li>Adjust the temperature of the cryostat to -26 &#176;C and wait until it has stabilized. Maintain the frozen tissue block at -26 &#176;C throughout the entire sectioning procedure. Absolutely avoid thawing the tissue at any time during the procedure.</li>
	<li>Cool the razor blade, the cutting support, the anti-roll device and the brush to -26 &#176;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.&#160;</li>
	<li>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.&#160; 
	Note: Using positively charged glass slides will greatly favor the adhesion of fresh frozen tissue sections due to higher electrostatic attraction.</li>
	<li>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.</li>
	<li>Place the wet mount in the disc holder of the cryostat. Adjust the cut thickness to 5-6 &#181;m 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.&#160;</li>
	<li>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.&#160;</li>
	<li>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.&#160;
	<ol>
		<li>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.&#160;</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.).</li>
</ol>
3. Indirect Immunofluorescence
<ol>
	<li>Locating sections
	<ol>
		<li>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.&#160; 
		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.&#160;</li>
		<li>Rehydrate tissue sections by covering them with a drop of ~250 &#181;l of PBS for a few minutes at RT. Fix tissue sections by covering them with ~250 &#181;l of a freshly prepared 3% PFA solution in PBS for 10 to 15 min.&#160; 
		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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.&#160;</li>
		<li>Preheat the AR solution to 95 &#176;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 &#176;C.&#160;</li>
		<li>Remove the beaker from the water bath and leave the glass slides for another 10 min in the buffer.</li>
	</ol>
	</li>
	<li>Immunodetection
	<ol>
		<li>Rinse tissue sections with PBS (~250 &#181;l/section) and saturate them with a solution of 3% bovine serum albumin (BSA, ~250 &#181;l/section) in PBS for at least 30 min at RT.&#160;</li>
		<li>Put 30-50 &#181;l 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.</li>
		<li>Place the same volume of the diluent (2% BSA in PBS) alone on a tissue section to perform a negative control without primary antibody.
		<ol>
			<li>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).</li>
		</ol>
		</li>
		<li>Place the glass slides in a humidified box O/N at 4 &#176;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).&#160;</li>
		<li>Thoroughly wash tissue sections with PBS at least four times for 10 min at RT.</li>
		<li>Dilute the appropriate secondary antibody (rhodamine-conjugated goat anti-rabbit IgG (H + L), 1:300 dilution) in PBS containing 2% BSA, place 30-50 &#181;l of this solution on all tissue sections, and incubate for 1.5 hr at RT.
		<ol>
			<li>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).&#160;</li>
		</ol>
		</li>
		<li>Thoroughly wash the tissue sections with PBS at least four times for 10 min at RT.&#160;</li>
	</ol>
	</li>
	<li>Post-fixation (optional)
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Neutral lipids and DNA counterstaining
	<ol>
		<li>To visualize CLDs and MFGs, color neutral lipids by incubating tissue sections in 30-50 &#181;l of a PBS solution containing 3 &#181;g/ml of bodipy 493/503 for 10 min at RT. Rapidly rinse tissue sections twice with PBS.</li>
		<li>Counterstain nuclear DNA with 30-50 &#181;l of a PBS solution containing 3 &#181;M 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.</li>
	</ol>
	</li>
	<li>Section mounting
	<ol>
		<li>Remove PBS and place a drop of mounting medium on each tissue section.&#160;</li>
		<li>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.</li>
		<li>Seal the cover slip to the glass slide with nail polish and store tissue sections at 4 &#176;C to prevent their exposure to light until observation.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Acquire all pictures, including those of the corresponding controls, in the same conditions (exposure and general settings) for each color channel.</li>
	<li>Conventional microscopy
	<ol>
		<li>Perform epifluorescence microscopy with a microscope equipped with standard filters for fluorescein isothiocyanate (FITC, green), rhodamine (red) and DAPI (blue) emissions, &#215;20 to &#215;63 (oil-immersion, NA 1.3) objectives and a DP50 imaging camera.</li>
	</ol>
	</li>
	<li>Confocal microscopy</li>
	<li>Perform confocal microscopy with a microscope equipped with the ZEN software, using &#215;20 to &#215;63 (oil-immersion, NA 1.4) objectives and the 488- and 568-nm excitation wavelengths of the laser.</li>
</ol>
5. Image Treatment
Note: All image post-treatments are performed using the ImageJ free software (http://imagej.nih.gov/ij/).
<ol>
	<li>Superimpose image (merge)
	<ol>
		<li>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).&#160;</li>
		<li>Generate the composite picture from grayscale or colored images by using the command &#8220;Merge channels&#8221; (Image/Color/Merge Channels) and then attributing a color to each channel.&#160;</li>
		<li>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 &#8220;Merge channels&#8221; (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).</li>
	</ol>
	</li>
	<li>Image stack Z projection
	<ol>
		<li>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 &#8220;Maximum Intensity&#8221; 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.</li>
	</ol>
	</li>
	<li>Image stack 3D projection
	<ol>
		<li>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.&#160;</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Image stack to movie conversion
	<ol>
		<li>Open an image stack (File/Open) and save it as a movie in .AVI format by using the command &#8220;AVI&#8221; (File/Save As/AVI).</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

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 experimental conditions, i.e., those generating a strong and specific signal for the antigen of interest. The variables that must be considered for experimental design and optimization are: (1) the type of antigen (species, expression levels, subcellular location); (2) the epitope type (sequence, conformation, putative post-translational modifications); (3) sample preparation (embedding in paraffin or for frozen sections); (4) the fixation method (perfusion or immersion); (5) the fixative used (formaldehyde, alcohol or acetone); (6) the blocking reagent used (normal serum, BSA or non-fat milk); (7) the AR step; (8) the detection method (direct or indirect); (8) the type of primary antibody (monoclonal or polyclonal); (9) the secondary antibody (species and label); (10) counterstains (nuclear and/or other cellular compartment labeling); and (11) the mounting medium (see Table 1 for details). The fixation and the blocking steps, at least, require the optimization of additional factors such as concentration, pH, temperature, incubation time and diluent.

The first crucial aspect concerns the preparation of tissue samples, which is closely linked to the fixation method, which in turn influences the quality of the results. For example, tissue pieces can be fixed or not prior to embedding. This step may also depend on the embedding method chosen, i.e., OCT compound vs. paraffin embedding, which itself sometimes depends on the primary antibody used. Tissue fixation can be performed in vivo by perfusing an anesthetized animal with a fixative solution. This method is useful to preserve antigens when studying intact tissues but may not be sufficient to fix the tissue of interest. In this case, small tissue pieces (no thicker than 10 mm) can be immersed in the fixative solution. Frozen tissue can be prepared by immersing the tissue in liquid nitrogen or isopentane, and snap-freezing is highly recommended for the subsequent detection of post-translational modifications such as phosphorylation. However, unlike paraffin-embedded tissue, freezing is not adequate for long-term preservation of the tissues due to the formation of ice crystals within the cells that may alter subcellular morphology. Once cut, frozen tissue sections can be stored at -80 &#176;C for up to 1 year. In any case, the preparation of the tissue samples is a compromise between preserving the tissue/cellular architecture and preserving the epitope integrity.

Since it alters the chemical composition of tissues, it is critical to optimize the fixation conditions to avoid both incomplete (under&#64257;xation) and excessive (overfixation) &#64257;xation.
Indeed, underfixation may reduce the specific signal by promoting the proteolytic degradation of certain antigens. On the other hand, overfixation can alter the specific labeling by masking the epitope or generating a strong non-specific background. Thus, in addition to the choice of the fixative solution, other parameters such as the incubation time, the temperature and the pH will affect tissue fixation. Although PFA is the most common fixative used for IHC, it cannot be considered as a &#8220;universal&#8221; fixative. PFA induces protein-protein and protein-nucleic acid cross-links and can thus artefactually modify the epitope (overfixation) and then prevent its recognition by the primary antibody. However, the epitope can be further unmasked by AR techniques (see below). PFA may also be unsuitable for the detection of certain antigens, as it has been shown to induce the translocation of some phosphorylated proteins from the membrane to the cytoplasm. In such cases, PFA must be replaced by appropriate alternative fixatives such as alcohol. Unlike PFA, alcohols such as methanol or ethanol do not mask epitopes since they allow tissue fixation by replacing water molecules in tissues. This can lead to the precipitation of the proteins and then prevent antibody/epitope interaction due to conformational changes. It is widely thought that alcohols do not penetrate and thus do not preserve tissue morphology as well as PFA. Acetone is another alternative fixative, which is commonly used when working with unfixed, snap-frozen tissue sections. However, acetone is a strong dehydrating agent and can lead to irreversible precipitation of tissue proteins.

For some antigens, an additional step of AR may be required to obtain a good signal, mainly if the fixative induces the conformational change or alters the electrostatic charge of the epitope (masking of the epitope). AR methods aim to reverse these processes to restore the immunoreactivity of the epitope and its subsequent interaction with the primary antibody. AR methods mainly rely on two approaches: (1) protease-induced epitope retrieval, i.e., with enzymes such as proteinase K, trypsin or pepsin, which cleave peptides that mask the epitope; and (2) heat-induced epitope retrieval, i.e., using a microwave oven, pressure cookers, vegetable steamers, autoclaves or water baths. This latter approach is especially time-, temperature-, buffer-, and pH-sensitive, and the optimal conditions must be empirically determined (an example is provided in the Protocol section). Alternatively, the affinity of an antibody for its antigen can be enhanced by changing the pH or the cation concentration of the antibody diluent.

A permeabilization step is sometimes required to obtain a good signal for an intracellular epitope in thick tissue sections, particularly for nuclear antigen staining. This can be achieved in various ways by using: (1) alcohols or acetone as fixatives; or (2) detergents such as Triton, NP-40 (0.1-0.2% in PBS, 10 min), digitonin, saponin or Tween 20 (0.2-0.5% for 10 to 30 min) after PFA fixation. However, the choice of the detergent depends on the cellular location of the epitope detected. Indeed, harsh detergents such as Triton-X100, which solubilize cellular membranes, are suitable for nuclear epitope detection but can lead to signal alteration upon the extraction of some membranous proteins. The use of milder detergents (saponin and Tween 20) are more suitable for the detection of cytoplasmic epitopes.

The second critical step is the blocking of non-specific staining. The binding of an antibody to its target epitope is governed by intermolecular forces (e.g., hydrophobic and ionic interactions, hydrogen bonding). Thus, interactions of primary and/or secondary antibodies with other proteins than their target antigens may result in non-specific staining. This generates high fluorescence background, which prevents the visualization of the protein of interest (low signal/noise ratio). Blocking reagents reduces non-specific interactions without impairing specific antibody/epitope interaction. A common procedure consists of incubating tissue sections with heat-inactivated normal serum or BSA. When using a normal serum, it must be from the same species as that of the host animal of the secondary antibody or from an unrelated species. In all cases, the selected blocking reagent must also be added to the diluents for the primary and secondary antibodies. Furthermore, the use of non-ionic detergents such as Triton X-100, Tween 20 or saponin helps to reduce non-specific interactions.

The third and probably most important parameter is the primary antibody selection and optimization. Obviously, the best choice is a high-quality antibody with minimal cross-reactivity. As monoclonal antibodies usually exhibit high affinity and specificity for a single epitope, they are the best tools to discriminate a particular member of a protein family with high sequence identity. However, the antibody/epitope interaction can be compromised if the target epitope has lost its native conformational state or when the access to the epitope is prevented by interactions with other proteins, post-translational modifications, temperature, pH, fixation and salt concentration. In such cases, polyclonal antibodies are more suitable as they recognize multiple epitopes of the same protein. Moreover, they are often more stable than monoclonal antibodies over a wide range of pH and salt concentration. Preliminary studies have to define the appropriate incubation conditions, i.e., working dilution (monoclonal antibody: 5-25 mg/ml, polyclonal antibody: 1.7-15 mg/ml), incubation time, diluent and temperature, which have to be empirically determined for each primary antibody. These parameters have to be optimized to determine the conditions that produce the optimal signal with low background noise. The specificity of the labeling is favored by longer incubation times at lower temperatures (i.e., 4 &#176;C vs. RT).

The choice to perform direct or indirect detection often depends on the level of antigen expression. For example, a highly expressed epitope can simply be detected with a fluorochrome-conjugated primary antibody, thus allowing a fast and simple multicolor staining while avoiding possible non-specific background due to the use of a secondary antibody. However, direct IF may generates a low signal at a higher cost, and can sometimes be difficult, when labeled antibody are not commercially available. Conversely, IIF is more sensitive to detect lower expressed epitopes as the generated signal is more intense due to the interaction of at least two labeled secondary antibodies (raised against the primary antibody host species) with the primary antibody (amplification). Furthermore, a wide range of secondary antibodies conjugated to various fluorophores are commercially available, relatively inexpensive, and quality controlled. However, this approach can induce cross-reactivity and thus requires to carefully choose primary antibodies that are not produced in the same species or of different isotypes when performing multiple-labeling experiments. IIF also sometimes requires extra blocking steps and must include systematic negative controls (see below). Amplification can be further achieved by using a biotin-conjugated secondary antibody and fluorescently labeled avidin or streptavidin (four biotins bound per molecule). Nevertheless, this amplification method requires additional steps to prevent non-specific binding and may not be adapted for the staining of some tissues (liver, kidney, heart, brain, lung and lactating mammary gland) due to the presence of high levels of endogenous biotin. However, endogenous biotin can be blocked by pre-incubating the sample with avidin and subsequently with biotin before incubation with the primary antibody. The choice of the conjugated fluorochromes, which are small chemical molecules with the property to emit light when excited by light of a shorter wavelength, primarily depends on the type of microscope equipment available.

When properly designed to limit both cross-reactivity between the antibodies and crossover between the spectral properties of the fluorochromes used, immunofluorescence-based IHC allows the simultaneous visualization of multiple cellular targets.

The last critical point regarding IHC experiments concerns the positive and negative controls that must be performed to support the validity of staining, to identify experimental artifacts and for the accurate interpretation of the results. Some tissues exhibit high fluorescent background (referred to as autofluorescence) that could lead to a misinterpretation of the results. Thus, tissue sections have to be observed under both fluorescence and bright-field illumination before starting the IHC experiment. A negative control that omits the primary antibody must systematically be included in each IHC experiment in order to ensure that a potential non-specific binding of the secondary antibody is negligible and does not obscure or resemble the specific staining pattern. An isotype control can be performed when working with a monoclonal primary antibody by replacing it with a non-immune antibody of the same isotype (e.g., IgG1, IgG2A, IgG2B, IgM) at the same concentration. This control helps to estimate the non-specific staining, which may be due to the interactions of antibodies with the sample. To demonstrate the specific binding of an antibody to its antigen, an absorption control can be achieved in two ways by pre-incubating the primary antibody: (1) with its soluble immunogen (10:1 molar ratio) O/N at 4 &#176;C; and (2) with cells or tissue sections that express the epitope of interest but that differ from the studied tissue (for example, see Figure 4B in59). In both cases, the consequent depletion of the primary antibody should lead to little or no staining. Another type of control can be done using an irrelevant primary antibody, i.e., directed against an epitope that exhibits a cellular localization that is different from the epitope of interest (nuclear vs. cytoplasmic). The irrelevant antibody must be of the same isotype and species as the primary antibody of interest. Additional controls for IHC experiments can include the use of samples from tissues known to express (transgenic animals) or not (knock-out animals) the epitope of interest. This can provide a useful reference and help to optimize the IHC procedure.

A main limitation of IF techniques is that they can only be applied to fixed (dead) and/or permeabilized cells, both procedure potentially inducing artifacts. Other limitations of this approach are due to the use of a microscope for the observation of the samples. First, as the optical resolution of epifluorescence and confocal microscopes is limited, location or co-location of the detected proteins should not be over-interpreted. Secondly, photobleaching, i.e. fading of the fluorescence intensity over time when exposed to light, is essentially due to generation of reactive oxygen species in the sample upon fluorescence excitation which, in turn, leads to the photochemical destruction of the fluorophore. Photobleaching can be reduced by: a) keeping the samples protected from light during the IF experiment and storage until their observation; b) using an antifade agent (reactive oxygen species scavengers) in the mounting medium; c) reducing the intensity and/or duration of the excitation light; d) increasing the concentration of fluorophores or using a low concentration of a fluorochrome with high-quantum efficiency; and e) using robust fluorophores that are less prone to photobleaching (i.e. Alexa Fluors, Seta Fluors, or DyLightFluors). Thirdly, autofluorescence is often due to the presence of flavin coenzymes (FAD and FMN: absorption, 450 nm; emission, 515 nm) and reduced pyridine nucleotides (NADH: absorption, 340 nm; emission, 460 nm) in mammalian cells. Furthermore, the use of aldehydes, particularly glutaraldehyde, to fix the samples, can result in high levels of autofluorescence. This can be minimized by washing the samples with 0.1% sodium borohydride in PBS prior to antibody incubation and/or by selecting probes and optical filters that maximize the fluorescence signal relative to the autofluorescence. Fourthly, fluorescence overlap (also termed bleed-through, crossover or crosstalk) is mainly due to the emission spectral properties of the fluorophores as they often exhibit very broad bandwidths, different, asymmetrical spectral profiles, as well as various peak emission wavelengths and number of maxima. Fluorescence overlap occurs when working with several fluorophores (multicolor labeling) and is characterized by the emission of one fluorophore in the channel (filter) of another fluorophore. Bleed-through artifacts must be minimized as they often complicate the interpretation of IF results, particularly in the case of co-localization or quantitative studies. As balancing of the fluorophores emission can only be slightly improved by the IF procedure, bleed-through can mainly be reduced at time of image acquisition by using an optimized fluorescence filter sets and/or photomultiplier detector in order to properly separate the spectral profiles of the fluorophores. In this regard, confocal microscopy is well-suited to multicolor imaging as it allows the differentiation fluorescence emission spectra of individual fluorophores by directing each signal to a particular detection channel. Moreover, confocal microscopy allows to adjust the gain, photomultiplier voltage, or laser power for the individual detection channels for the sequential acquisition (only one fluorophore at a time) of the labeling. Ideally, single-label controls must be performed to quantify the bleed-through and eventually remove it computationally. A control without secondary antibodies (background control) can be prepared to set the limits of signal gain and offset of each channel for optimal image acquisition. It can also be used for post-acquisition processing to correct image background (autofluorescence).

In conclusion, the method described provides a simple standard protocol for easy realization of immunostaining on mammary gland sections. Nevertheless, the principal steps of an IHC experiment must be optimized for each antigen/antibody couple in order to visualize specific staining and to minimize non-specific background signals. The described method also includes several basic methods for the post-treatment of most of the images obtained. Fluorescence-based immunodetection is a powerful method with a wide range of applications from the cellular localization of an antigen to diagnosis. New advances in these approaches will be achieved with the future development of new fluorophores, acquisition devices and microscopy techniques, to image previously unobserved details of biological structures and processes.

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 development1-5.
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 puberty6-9.
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.&#160;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 epithelium11-13.&#160;
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 development19-21. 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.&#160;
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 lactation9,22,23, 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 laminin24,25, and suppress apoptosis in MESCs26,27. 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).&#160;
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 proximity30,32. 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) membrane38-41. 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 &#181;m in diameter) by budding, being enwrapped by the MESC apical plasma membrane40-42. 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:756px;" width="755">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Dissection</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:236px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Pins</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ethanol</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Scissors</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel and adapted blades</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ice</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Towel paper</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Tissue sample preparation</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Phosphate Buffered Saline (pH7.4)</td>
			<td style="width:155px;">Sigma</td>
			<td>P-3813</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Paraformaldehyde (PFA, 32% EM grade, 100 ml)</td>
			<td style="width:155px;">Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>personnal protection equipment required WARNING: this product will expose you to Formaldehyde Gas, a chemical known to cause cancer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT compound/Tissue Tek</td>
			<td style="width:155px;">Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose (D-saccharose)</td>
			<td style="width:155px;">VWR</td>
			<td>27480.294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plastic molds</td>
			<td style="width:155px;">Dominique Dutscher</td>
			<td>39910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Liquid nitrogen</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Cryostat/sample support</td>
			<td style="width:155px;">Leica&#160;</td>
			<td>CM3050S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Razor blades (SEC35)</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td>152200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Slide box</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Glass slides Superfrost/Superfrost Ultra Plus</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td>10143560W90/1014356190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Brushes</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">IHC</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Super Pap Pen</td>
			<td style="width:155px;">Sigma</td>
			<td>Z377821-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Permanent marker (black)</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:239px;">50 mM NH4Cl in PBS</td>
			<td style="width:155px;">Sigma</td>
			<td>A-0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">0.1 M glycine in PBS</td>
			<td style="width:155px;">VWR</td>
			<td>24403.367</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Antigen Retrieval solution: Tris 100 mM 5% urea pH9.6</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Heater (up to 100&#176;C)</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:155px;">Sigma</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Vectashield (anti-fading mounting medium) without DAPI/with DAPI</td>
			<td style="width:155px;">Vector Laboratories</td>
			<td>H-1000/H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Glass coverslips 22x50mm (microscopy grade)</td>
			<td style="width:155px;">VWR</td>
			<td>CORN2980-225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nail polish</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Primary antibodies</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:239px;">Rabbit anti-mouse caseins (#7781; 1:50 dilution)</td>
			<td style="width:155px;">generously gifted by M.C. Neville (University of Colorado Health Sciences 
			Center, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Mouse anti-cytokeratin 8 (CK8, clone 1E8, 1:50 dilution)</td>
			<td style="width:155px;">Biolegend (Covance)</td>
			<td>MMS-162P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Mouse anti-cytokeratin 14 (CK14, cloneLL002, 1:50 dilution)</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td>MS-115-P0/P1</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:239px;">Rabbit anti-butyrophilin (1:300 dilution)</td>
			<td style="width:155px;">generously gifted by I.H. Mather (Department of Animal and Avian Sciences University of Maryland College Park, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:239px;">Rabbit anti-Stx6 (1:50 dilution)</td>
			<td style="width:155px;">generously gifted S. Tooze 
			(Cancer Research UK, London Research Institute, London, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Rabbit anti-VAMP4 (1:50 dilution)</td>
			<td style="width:155px;">Abcam</td>
			<td>ab3348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Secondary antibodies</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;">Rhodamine-conjugated goat anti-rabbit IgG (H + L) (1:300 dilution)</td>
			<td style="width:155px;">Jackson ImmunoResearch Laboratories</td>
			<td>111-025-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Counterstains</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Bodipy 493/503</td>
			<td style="width:155px;">Life Technologies (Molecular Probes)</td>
			<td>D-3922</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">DAPI (4-6-diamidino-2-phenylindole)</td>
			<td style="width:155px;">Life Technologies (Molecular Probes)</td>
			<td>D-1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Observation/Image capture</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:239px;">conventional fluorescence microscope</td>
			<td style="width:155px;">Leica Leitz DMRB 
			microscope</td>
			<td></td>
			<td style="width:236px;">Standard filters for FITC, Rhodamine 
			and DAPI emissions,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#215;63 oil-immersion objective (NA 1.3), DP50 imaging camera (Olympus), Cell&#710;F software (Olympus)</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:239px;">Laser Scanning Microscope (confocal microscopy)</td>
			<td style="width:155px;">Zeiss LSM 
			510 microscope</td>
			<td></td>
			<td style="width:236px;">Plan-Apochromat &#215;63 oil-immersion objective (NA 1.4),&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; CLSM 510 software,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Confocal facilities, MIMA2 Platform, INRA Jouy-en-Josas, France, http://mima2.jouy.inra.fr/mima2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Image treatment</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ 1.49k software</td>
			<td style="width:155px;"></td>
			<td></td>
			<td>Free software</td>
		</tr>
	</tbody>
</table>

indirect immunofluorescence on frozen sections of mouse mammary gland

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.

Watch this Scientific Journal Video about Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland at JoVE.com

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

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.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

indirect-immunofluorescence-on-frozen-sections-of-mouse-mammary-gland

Nanyang Technological University

Research

JoVE Journal

Biology

36.7K Views.  INRA. 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.

Video: Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...