For immunohistochemical analysis, after warming the sections for 20 minutes on the hot plate, wash the slides three times in PBS for 10 minutes per wash, and eliminate the endogenous peroxidase with a 10-minute 3% hydrogen peroxide incubation.
At the end of the incubation, wash the samples three times in PBS as demonstrated, and block any nonspecific binding with blocking buffer, for at least one hour at room temperature. Then, replace the blocking buffer with the primary antibody cocktail of interest for an overnight incubation at 4 degrees Celsius.
The next morning, aspirate the excess primary antibody solution, and wash the slides three times in PBS. After the last wash, incubate the slides with the appropriate biotinylated secondary antibody cocktail followed by labeling with streptavidin horseradish peroxidase complex at room temperature.
To visualize the immunoreactive cells, label the samples with DAB until a light or dark brown color develops and counterstain the sections with hematoxylin and 0.3% diluted ammonia.
When the slides have dried, use a light microscope to obtain brightfield images of the tissue sections under a 10 times magnification, and use an image processing program to identify and quantify the population of the marked immune cells in both the epithelium and the lamina propria.
