Peroxidase staining of unlabelled rat IgG antibodies. Staining procedure All steps are performed at room temperature. 1 Cover tissue sections with normal serum from the same animal species that produced thesecondary antibody (step 4), undiluted or in a dilution with PBS up to 1: 20. Incubate 10 minutes and drain. Wash once with PBS. 2 Cover section with primary rat antiserum diluted in PBS and incubate 30-60 minutes. It may be necessary to incubate overnight in a moist chamber if tissue antigen is limiting or if greater penetration is required to increase antigen detection signal. Drain and wash gently by flooding section with PBS. Incubate 5-10 minutes. Repeat wash two times at 5-10 minutes intervals. 3 Repeat the first step if persistent background is encountered in preliminary trials. 4 Cover tissues section for 30-60 minutes with secondary anti rat IgG (H+L) antiserum, diluted with PBS to 1:50 to 1:100. The secondary antiserum is used in excess to ensure free antigen-binding sites to bind the PAP complex. Drain and wash gently by flooding section with PBS. Incubate 5-10 minutes and drain. Repeat wash two times at 5-10 minutes intervals. 5 Make a solution of the R/PAP of 25-50 µ g/ml with PBS containing 2% normal serum of the same host as the secondary antiserum. Incubate 30-90 minutes, drain and wash gently by flooding section with PBS. Incubate 5-10 minutes and drain. Repeat wash two times at 5-10 minutes intervals. 6 Cover section with 0.05 M Tris-HCl, pH 7.6 containing 0.015% H 2 O 2 and 0.05% diamino- bezidinetetrahydrochloride. Incubate for 30-60 minutes, dehydrate and mount for microscopy. If residual endogenous peroxidase activity is encountered, cover tissue section with 0.5-3.0% H 2 O 2 in absolute methanol for 5 minutes, drain off H 2 O 2 and gently flood section with PBS. Incubate 5 minutes, drain and repeat with fresh buffer.