Encyclopedia of Experiments: Immunology
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For antigen retrieval, transfer the rack into a glass container with 200 microliters of 95 degrees Celsius, 10 millimolar per liter, citrate buffer for 30 minutes. At the end of the incubation, allow the slides to cool to room temperature for 20 minutes before rinsing with running water for five minutes.
To remove the endogenous peroxidase activity, first, use a hydrophobic marker to draw a circle around each tissue sample, before incubating the samples in 200 microliters of 3% hydrogen peroxide for 10 minutes, followed by three washes in 200 microliters of Tris-buffered saline solution or TBSS per wash. After the last wash, dab each slide gently to remove any excess buffer, and add 200 microliters of the primary antibody cocktail of interest and a coverslip to each slide for a one-hour incubation in a humid chamber at room temperature.
At the end of the incubation, discard the coverslips and rinse the slides with three two-minute washes in fresh TBSS. After removing the excess buffer, add 200 microliters of an appropriate biotinylated secondary antibody solution and a coverslip to each slide for a 45-minute incubation in the humid chamber at room temperature. After discarding the coverslips, rinse the slides with TBSS, as demonstrated. Remove any excess buffer.
Enable the sections with 200 microliters of DAB substrate buffer for 10 minutes at room temperature. At the end of the incubation, wash the slides with TBSS and running tap water for 10 minutes, before counter-staining with hematoxylin for three minutes. Then, add 70 microliters of an appropriate mounting medium to each slide, and slowly tip a glass coverslip onto the mounting medium to avoid creating bubbles as the coverslip is lowered into place.
For histopathologic analysis, place the slide onto the stage of a light microscope and assess the vascular architecture of the first tissue section. Then, quantify the number of macrophages relative to the number of lymphocytes per section.
