Ideally, single-label controls must be performed to quantify the bleed-through and eventually remove it computationally. 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. i.e.Jouy-en-Josas/AgroParisTech). 1. Mammary Gland Sample Preparation Mouse mammary gland dissection Euthanize mice at day 10 of lactation by cervical dislocation and pin the animal down with its abdomen facing up.? Wet the ventral area with ethanol and dry it with a paper towel.? 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 around the mouse. Pull the Indolelactic acid skin away from the peritoneum and pin down one side of the skin at a time, stretching it taught.? 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 Indolelactic acid be useful to analyze the global morphology of the mammary gland under various conditions (physiological developmental stages, diseases, in vivo treatments). Remove the lymph node located at the junction of the abdominal and the inguinal glands44. Mammary tissue fixation 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.? Quickly dry the fragments on a paper towel and put them in a cold PBS solution made up of 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 LRP1 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). Sucrose infusion Quickly rinse the mammary fragments in cold PBS and immerse them in a cold PBS solution made up of 40% sucrose (D-saccharose, C12H22O11, Mr 342.3 g/mol) for 16 to 48 hr at 4 C under gentle shaking. 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.? 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.? 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. 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 usually often required for fat or lipid-rich tissues such as virgin mammary gland. 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. Completely avoid thawing the tissue at any time during the procedure. 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.? 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 Indolelactic acid attraction. Cover the surface of a metal tissue disc with OCT compound.