Staining Protocols for Lymphoid Tissue:

Procedure:

• Dry the sections for 2 hours at 37°C on a slide warmer. This must be done whether the slides have been stored at 4°C for some time or just collected.
• Pretreat the sections with trypsin at 37°C. Trypsin concentrations must be determined separately for each antibody. Use trypsin solution that has been preheated at 37°C for 30 minutes. Cover section with at least 100 microliters of solution.
• Wash in PBS for 10 minutes at room temperature. Refresh the buffer 4 to 5 times.
• Preincubate in normal serum from the animal species in which the second antibody is raised for 30 minutes at 37°C. Only perform this step if aspecific background is present using a particular antibody.
• Drip off excess serum and apply the first antibody in an appropriate concentration and incubate for 2 hours at 37°C.
• Wash in PBS for 10 minutes at room temperature. Refresh the buffer 4 to 5 times.
• Block endogenous peroxidase in a solution of 0.06% hydrogen peroxide in phosphate buffered saline, pH 7.4, for 30 minutes at room temperature.
• Wash in PBS for 10 minutes at room temperature. Refresh the buffer 4 to 5 times.
• Incubate in appropriate dilutions of the secondary antibody, containing 5% normal serum for 60 minutes at room temperature.
• Wash in PBS for 10 minutes at room temperature. Refresh the buffer 4 to 5 times.
• Develop the peroxidase activity in daminobenzidine (DAB).
• Counterstain the sections in hematoxylin or, if a more advanced morphological detail is necessary, in periodic-acid-Schiff reagent.
• Cover with glycerin-gelatin and cover glass