Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

The overall goal of this procedure is to localize proteins of interest in the mammary tissue of the mouse during lactation. This method can help answer key questions in the cell biology field, such as where the snap proteins involved in mid product secretion, localized in the mouse, mammary epithelial cells during lactation. The main advantage of this technique is that you can rapidly and easily observe the location of proteins in a given tissue context.

Following euthanasia position the mouse on its back wet the ventral area with ethanol and then dry it with a paper towel. Next, use forceps to pull up the abdominal skin between the two hind legs and make a one centimeter long incision through the skin with a pair of sharp scissors. Starting from this first incision, cut the skin up to the neck and then pull the skin away from the peritoneum.

Pin down one side of the skin at a time, stretching each side taut. Next, use a sterile swab to push the abdominal and the inguinal mammary glands away from the skin and dissect them away from the peritoneum. Place the dissected mammary tissue onto a dissection plate cooled by ice.

Finally, remove the lymph node located at the junction of the abdominal and the inguinal glands Using a scalpel. Cut the memory tissue into fragments approximately three cubic millimeters in size, and immediately rinse these fragments in PBS. In order to remove as much milk as possible, quickly dry the fragments on a paper towel and then put them in an ice cold PBS solution containing 4%para formaldehyde for 10 to 15 minutes on ice.

To gently fix the tissue following fixation, quickly rinse the mammary fragments in ice cold PBS, and then immerse the fragments in ice cold PBS containing 40%sucrose for 16 to 48 hours at four degrees Celsius. While gently shaking proper properly, label the plastic cryo molds and then fill them a third of the way up with optimal cutting temperature compound at room temperature. Place one fragment containing two to three cubic millimeters of mammary tissue into each mold and cover them with additional compound.

Next, freeze the optimal cutting temperature compound and the sample by placing the molds at the surface of a liquid nitrogen bath until the entire mold becomes opaque. Store the blocks in the freezer at minus 80 degrees Celsius until the samples are sectioned. Adjust the temperature of the cryostat to minus 26 degrees Celsius and wait until the temperature stabilizes.

In the meantime, place the blocks to be sectioned in the cryostat so that they can equilibrate to minus 26 degrees Celsius. As well. Keep the frozen tissue block frozen throughout the entire sectioning procedure.

Additionally, cool the razor blade, the cutting. Support the anti roll device and the brush to minus 26 degrees Celsius by placing them in the cryostat for at least 10 minutes. Also, place a slide box inside the cryostat in order to be able to store glass slides as the sections are made.

While these items cool, properly label the positively charged glass slides that will be used to collect the tissue sections and to maintain them at room temperature. Next, remove a sample from its mold. Cover the surface of a metal tissue disc with room temperature optimal cutting temperature compound, and push the frozen sample onto it.

Place the wet mount inside the cryostat and let it cool for at least 15 minutes. Once cooled, place the wet mount in the disc holder of the cryostat and adjust the cut thickness to between five and six microns. Next, adjust the position of the anti-roll device by making cuts of the mounting medium until the slices form evenly and correctly.

Once the settings are correct, perform tissue sections by turning the wheel in a continuous uniform motion. Use a brush to grab and maneuver the section across the stage. Retrieve the tissue sections one by one by holding the glass light above the section and slowly angling it down until the tissue section touches the slide.

Using a hydrophobic barrier pen, draw a circle around the slide mounted tissue. Let the circle dry for approximately one minute at room temperature. Then use a permanent marker to draw a line around the tissue sections on the backside of the slide.

Next, we hydrate the tissue sections by covering them with a drop of PBS for a few minutes at room temperature. Then fix the tissue sections by covering them with freshly prepared 3%PARAFORM aldehyde solution in PBS for 10 to 15 minutes. Then permeate them in PBS with 0.05%saponin for 10 to 15 minutes.

Next, incubate them in PBS with 3%bovine serum albumin for at least 30 minutes at room temperature following antigen retrieval When needed, rinse the tissue sections with PBS and then incubate them in PBS with 3%of serum albumin for at least 30 minutes at room temperature. Next, remove the blocking solution and at 30 to 50 microliters of primary antibody diluted in PBS containing 2%BSA, so that each tissue section is completely covered. Place the same volume of the diluent alone on a tissue section to perform a negative control.

Then place the glass slides in a humidified box overnight at four degrees Celsius the next day. Thoroughly wash the tissue sections with room temperature PBS for 10 minutes and repeat at least four times. Dilute the appropriate secondary antibody in PBS containing 2%BSA in accordance with the manufacturer's instructions, and then place 30 to 50 microliters of this solution on all of the tissue sections.

Incubate the sections for one and a half hours at room temperature. Then thoroughly wash the tissue sections again with room temperature PBS for 10 minutes and repeat at least four times. Next, color the neutral lipids in the sections by incubating the tissue in 30 to 50 microliters of A PBS solution containing three micrograms per milliliter of boda P 4 93, 5 0 3 for 10 minutes at room temperature.

Following incubation rapidly rinse the tissue sections twice with PBS counterstain, the nuclear DNA with 30 to 50 microliters of a PBS solution containing three micromolar DPI for 10 minutes at room temperature. Wash the tissue sections twice with PBS before mounting the slides. For observation, remove PBS and place a drop of mounting medium on each tissue section.

Then place one side of a cover slip at an angle against the slide making contact with the outer edge of the liquid drop, and then lower the cover slowly avoiding air bubbles. Allow the liquid to spread between the glass slide and the cover slip for a few minutes, and then remove the excess mounting medium with a paper towel. Seal the cover slip to the glass slide with nail polish and store the tissue sections at four degree Celsius.

Finally, image the sections as described in the accompanying text protocol. These images show the localization of caines in the lactating mouse mammary gland. At day 10 of lactation in the presence or in the absence of pups during suckling, caines appear to be mostly accumulated in the apical region.

In addition, confocal microscopy reveals that caines are also present, although to a lesser extent, at the basal side of epithelial secretory cells in the presence of pups when milk secretion is slowed down, caines also appear accumulated beneath the apical plasma membrane and are clearly observed at the basal side of memory. Epithelial secretory cells butyrophilin shown in red is one of the major proteins associated with milk, fat, globules, and milk. This transmembrane protein is mainly localized at the apical plasma membrane of the mammary epithelial secretory cells, and is consequently found at the surface of the milk fat globules after it's released by budding Unmastered.

Visiting can be done in eight to 10 hours if it is performed properly. While attempting this procedure, it is important to remember to keep away from light during the immuno detection procedure Following this procedure. Other methods like ImmunoGold detection using transmission electron microscopy can be performed in order to answer additional questions like the subcellular localization of the protein of interest after its development.

This technique paved the way for researchers in the field of cell biology to explore proteins localization in normal and pathological tissues in various species. After watching this video, you should have a good understanding of how to help serve the tissue distribution of a protein of interest. Don't forget that working with animals, regions and some instrumentation can be extremely hazardous, and precautions such as wearing gloves and working under an extractor hood should always be taken while performing this procedure.