Immunofluorescence is a basic method that can help answer key questions in the adrenal field about adrenal zonation, cell identity and function. The main advantage of this technique is that it allows the study of multiple proteins while preserving the morphology of the adrenal tissue. To remove the murine adrenal gland, cut around the surrounding adipose tissue and place the gland in one well of a 24-multiwell plate containing fresh PBS on ice.
Transfer the gland onto a Petri dish or glass support under a dissecting microscope and use two 25 gauge needles to remove the adjacent fat tissue. When all of the fat has been removed, fix the tissue in 4%paraformaldehyde at four degrees Celsius with rocking for two hours. At the end of the incubation, rinse the gland with three 15-minute washes at four degrees Celsius in one to two milliliters of fresh PBS per wash.
After the last wash, dehydrate the gland in two sequential ethanol immersions at four degrees Celsius with rocking for two hours per incubation. To prepare the adrenal tissue for processing, wrap the gland in gauze and enclose the adrenal in a tissue-embedding cassette. Then, place the cassette in a jar filled with 70%ethanol for four degrees Celsius storage until processing.
To process the tissue, insert the cassette into the tissue processor for processing as indicated in the table. At the end of the program, transfer the cassette into an embedding station filled with melted 65 degree paraffin and use a pair of forceps to unwrap the gauze. Gently transfer the adrenal in the appropriate position at the center of the base of the embedding mold and pour the melted paraffin onto the gland.
Then, carefully move the embedding mold to a cooling tray to facilitate hardening of the paraffin. Before sectioning by rotary microtome, label a series of microscope slides and lay the slides on a 37 degree Celsius slide warmer. Dispense about 450 microliters of autoclaved type one ultra pure water onto each slide and set the section cutting thickness of the microtome to five micrometers.
Load the embedded tissue onto the cutting block and lower the paraffin block until the sample is at face level with the edge of the microtome blade. Rotating the hand wheel with a steady rhythm, perform an initial trimming to remove the paraffin and expose the adrenal tissue. Then, acquire five micrometer sections through the end of the adrenal gland using forceps to transfer each section into the water droplet onto one of the pre-labeled slides as it is collected.
After confirming the presence of tissue in the sections by microscopy, dry the slides on the hot plate for about two hours and bake them at least overnight on a slide tray at 37 degrees Celsius until no more water is visible. Once dry, store the slides in a slide box at room temperature. For immunofluorescence antigen retrieval, de-paraffinize the slides with two five-minute 100%xylene immersions, followed by rehydration in a descending ethanol and water immersion series.
After the water immersion, transfer the slides to a metal slide holder and place the holder in a beaker of boiling 0.01 molar citrate on a hot plate for 10 minutes. At the end of the incubation, remove the beaker from the hot plate and let it cool for 20 minutes, taking care that all of the adrenal sections are still covered with buffer. When the beaker is cooled, transfer the slides into a Coplin jar for three 10-minute washes in fresh PBS with gentle rocking.
After the last wash, carefully wipe the surface of each slide without damaging the tissue sections and use a hydrophobic marker to make a circle around each section. Place the slides in a humidified chamber and quickly add at least 50 microliters of blocking solution to each section for a one-hour incubation at room temperature. At the end of the blocking incubation, wash the slides three times in fresh PBS for five minutes per wash with rocking and return slides to the chamber.
Next, add the primary antibody cocktail of interest to each section for an overnight incubation at four degrees Celsius. The next morning, wash the samples with three 15-minute washes in fresh PBS per wash and label the slides with the appropriate secondary antibody solution for one hour protected from light. At the end of the incubation, wash the samples with three 15-minute washes on a rocker protected from light, labeling the nuclei of the sections with DAPI for seven minutes at room temperature protected from light after the last wash.
After three five-minute PBS washes with rocking, add 60 microliters of mounting agent suitable for immunofluorescence along the surface of one cover glass per sample and lightly press each slide tissue section side down onto a cover glass, taking care to avoid bubbles. Then, lay each slide flat in a dark container for curing at room temperature for at least 24 hours before imaging. During the fat removal process, it is critical not to let the adrenal gland dry out as dehydration can damage the tissue structure.
Following a successful removal of the surrounding adipose tissue, the adrenal gland should be easily detectable with no extra visible fat. Immunostaining of adrenal gland sections as demonstrated reveals nuclear steroidogenic factor one staining of the adrenocortical cells, tyrosine hydroxylase staining of the medullary cells, DAPI staining of the nuclei of non-steroidogenic cells of the outer capsule, and co-staining with adrenocortical and medullary cells. When attempting this procedure, it is important to remember that mouse adrenals are small and that their positions are somewhat variable making their detection difficult.
Therefore, it is crucial to have a clear view of the area during the dissection. These procedures are basic techniques for the study of the adrenal gland in vivo. Following these, additional methods of immunostaining can be performed to meet specific scientific needs.
After its development, immunostaining paved the way for researchers in the biomedical science fields to explore in situ protein expression, a useful tool in many disciplines and fields.
