- Begin by placing a thin tissue section in an ammonia solution to allow gentle antigen retrieval. Transfer the section to a polylysine-coated slide. Negatively charged cells bind to the positively charged polylysine. Next, add hydrogen peroxide to block the endogenous peroxidase activity. Wash the slide in Tris-Buffered Saline, TBS.
TBS promotes access of the antibodies to antigens by breaking the protein crosslinks. Wash the section with a block solution to reduce the non-specific antibody binding. Add primary antibodies to the section and incubate overnight. The primary antibody binds to its specific antigen present on the cell surface. Wash the slide with TBS.
Add Biotin conjugated secondary antibodies that attach to the Fc region of the primary antibody. Wash the section with streptavidin-biotin peroxidase enzyme. Next, add chromogenic substrate of peroxidase to the tissue. Peroxidase converts chromogen to a colored insoluble product.
Counterstain the tissue with hematoxylin solution. Hematoxylin binds to the acidic components of cells and turns them blue. Observe the tissue under a light microscope. The antigens are detected as red while the cells appear blue due to hematoxylin. In the following protocol, we will perform immunohistochemical staining to identify CD45 and CD1a cells from endobronchial biopsies.
- Before sectioning the embedded biopsy tissue, carefully adjust the knife holder in the angle of the biopsy block so the blade rests flat against the surface of the block. Then slowly cut at least two 2 micron thick sections from each endobronchial biopsy sample, using forceps to transfer each freshly cut tissue slice into a 0.05% ammonia and distilled water solution. After 45 to 90 seconds, use a poly-l-lysine coated microscope slide to pick up each unfolded section and allow the sections to dry for at least an hour.
When the sections have dried, label the slides as appropriate for the experiment and draw a hydrophobic barrier around each sample. Next, apply 1 milliliter of freshly prepared peroxidase block solution to the sections. After 30 minutes, remove the peroxidase block solution and then wash the sections three times for five minutes with TBS, then remove the TBS and add a 1% BSA blocking solution for another 30 minute incubation to block any non-specific antibody binding.
At the end of the incubation, add the primary antibody of interest, diluted in TBS, and cover slip the samples for an overnight incubation at room temperature. The next morning, wash the slides three times in TBS and apply the secondary antibody of interest for two hours at room temperature. At the end of the incubation, wash the slides three more times in fresh TBS and incubate the samples in freshly prepared streptavidin-biotin peroxidase complex solution for two hours at room temperature followed by three more TBS washes.
Then treat the dishes with freshly prepared AEC peroxidase substrate solution for 20 to 30 minutes. When the desired stain intensity develops, rinse the slides in running tap water for five minutes and counterstain the sections with filtered Mayer's hematoxylin solution. After two minutes, wash the slides in running tap water for another five minutes, then drain the slides and cover the sections with permanent aqueous mounting medium, drying the samples in an 80 degree Celsius drying oven.
After they have cooled, mount the samples with DPX mounting medium and a cover slip for their analysis at a 40 times magnification on a light microscope.