After a wash in water, slides were transferred into Tris-buffered saline (TBS; 0.05 M Tris and 0.85% NaCl, pH 7.6). Endogenous biotin was blocked using an avidin/biotin blocking kit (Vector Laboratories, Inc., Peterborough, U.K.) according to manufacturer’s instructions but incorporating a protein block of 5% BSA (Sigma) in TBS in the avidin step. Antibodies were diluted in 5% BSA/TBS (Table 1) and applied to the sections at 4°C overnight in a humidified chamber.
3,3-Diaminobenzidine Tetrahydrochloride Visualization
Sections were washed in TBS and then incubated for 30 min with the appropriate biotinylated (single-stain) or horseradish peroxidase (HRP; double-stain)-linked secondary antibody (DAKO, Ely, U.K.) diluted 1:500 in BSA/TBS. Following washes in TBS, single-stained sections were incubated with avidin-biotin-HRP complex (Vector Laboratories or DAKO) according to the manufacturer’s instructions. Bound antibody, on both single- and double-stained sections, was visualized using 3,3-diaminobenzi-dine tetrahydrochloride (DAB; DAKO). Negative controls, omitting the primary antisera, were included in each experiment (Figs. 1 and 2, insets).