The overall goal of this procedure is to target a specific protein in the rat tissue. This is accomplished by first isolating, fixing, and paraffin-embedding the tissue, followed by sectioning of the tissue and mounting it onto slides. The second step is to deparaffinize and rehydrate the paraffin-embedded tissue sections, and then block the endogenous peroxidase.
Next, the epitopes are uncovered by antigen retrieval. Subsequently, blocking is performed to reduce unspecific binding prior to introducing the antibodies. The final step is to detect the specific epitope by immunolabeling with primary and secondary antibodies, and to develop the signal from the labeled secondary antibody.
Ultimately, the signal is visualized using light microscopy to show the protein localization pattern in the tissue sections. Though this method can provide insight into the protein localization and expression patterns in the rat central nervous system and in the peripheral lymph nodes, it can also be applied to other organs and species. For demonstrating the procedure will be a technician.
Her name is Katalin Benedek, from the core facility of the microbiology tumor and cell biology center at the Karolinska Institutet. To begin tissue preparation, immerse the dissected brain, spinal cord, and peripheral lymph node tissue in four percent paraformaldehyde, and place it at four degrees Celsius for 24 hours. After the tissues are fixed, transfer the tissue into P-B-S and store it at four degrees Celsius until further processing.
To begin processing the tissue, use a razor blade to cut the tissue into approximately five-millimeter sized portions. Then, initiate the tissue hardening process with a vacuum infiltration processor. The standard procedure is based on submersion into ascending concentrations of ethanol, followed by xylol, and finally paraffin.
Next, place the tissue in a mold. Pour in the liquid paraffin around the sample to form a paraffin block, and store it at room temperature. Prior to sectioning the tissue, cool down the paraffin block overnight on the cold plate or in the refrigerator.
After the paraffin block has been cooled, place the block into the fixed holder of a sledge microtome which can move backwards and forwards across a knife. Then, adjust the block and the microtome knife to the optimal angle, which depends on the knife geometry and the cutting speed and technique. Cut three five-micron thick cross-sections from the paraffin block.
Then, transfer the cut sections into a water container and mount the sections from the water onto a commercial pre-coated adhesive glass slide. Next, press the mounted section carefully against a paper towel to remove residual water and potential air bubbles. Dry the mounted slides for a couple of hours in a stove set to 50 to 60 degrees Celsius.
Once the slides are dry, immerse the slides two times in xylol for 15 to 20 minutes each. Then, rinse each slide in 99 percent ethanol. To block the endogenous peroxidase activity, incubate the sections for 30 minutes in a methanol solution containing 0.25 percent hydrogen peroxide.
Then, continue rehydrating the tissue sections by rinsing with ethanol solutions containing increasing water content. Perform the final rehydration step in distilled water. Make sure to keep the sections moist until the cover slips are mounted.
To retrieve the antigen, heat the slides with a household steamer and EDTA buffer at pH 8.5 for 60 minutes. Then, cool down the slides at room temperature for approximately one hour. Once the slides are cool, rinse each slide three to five times in Tris-buffered saline, or T-B-S.
Next, incubate the tissue sections at room temperature for 30 minutes in a blocking solution containing 10 percent fetal calf serum and 90 percent wash buffer to block any non-specific background reactions. After the tissue sections have been blocked, dilute the required amount of primary antibodies in the blocking solution, and incubate the slides in this primary antibody solution overnight at four degrees Celsius. The next day, rinse the slides three to five times with T-B-S buffer.
Then incubate the slides with secondary antibodies diluted in the blocking solution for one hour at room temperature. Afterwards, again rinse the slides three to five times with T-B-S buffer. Then, incubate the slides with avidin horseradish peroxidase complex, diluted one to 100 in the blocking solution, for one hour at room temperature.
Once the one-hour incubation is complete, rinse the slides three to five times with T-B-S buffer. After preparing the fast blue solution as described in the text protocol, shake the dish lightly and filter the obtained mixture. Pour the solution onto the slides in the glass cuvette.
Begin the incubation at 37 degrees Celsius, and observe the developing process under the light microscope every 15 to 30 minutes. Once the incubation is complete, rinse the slides three to five times with T-B-S buffer, and then subsequently transfer the slides into P-B-S buffer. To make the D-A-B hydrogen peroxide developing solution, first dilute one milliliter of a D-A-B stock solution in 49 milliliters of P-B-S.
Add 16.5 microliters of hydrogen peroxide, and then filter the solution. Pour the solution onto the slides, and make sure to control the developing process under the light microscope in order to avoid high background that can mask the specific signal. Then, rinse the slides three to five times with P-B-S, followed by a final wash with distilled water or tap water.
Mount the slides with cover slips directly from the water, using aqueous mounting medium. Avoid creating air bubbles between the cover slip and the slide. After mounting the sections, incubate the slides overnight at four degrees Celsius to allow the mounting medium to dry completely.
Store the dried slides at room temperature. Double staining of peripheral lymph node tissue sections isolated from rats on day seven post-immunization revealed that cd8 positive t lymphocytes did not secrete the chemokine C-C-L 11. Alternatively, C-C-L 11 was found to co-localize with E-D one, a marker for lysosomal protein in activated macrophages.
I-H-C double staining of a rat's spinal cord identified C-C-L eleven production in neuronal perikarya, dendrites, and axons, but not in I-B-A one positive macrophages and brain-resident microglia cells. In addition, the majority of E-D one positive macrophages and brain-resident microglia cells were also found to not be C-C-L 11 positive. After having watched this video, you should have a good understanding of how to perform a specific and reliable immunohistochemical targeting of the protein directly in tissue sections.
