Light microscopy immunoperoxidase staining protocol

Select sections to process from the brain tissue bank.

Wash sections thoroughly with a phosphate-buffered saline(PBS, 0.01Molarity (M), 7.4) solution.

Treat the sections at Room temperature with a 1% sodium borohydride (NaBH4) solution in PBS for 0h 20m 0s.

Rinse sections thoroughly in PBS.

Pre-incubate sections for 1h 0m 0s at Room temperature in a solution containing 1% normal serum (from the species used to generate the secondary antibodies), 0.3% Triton X-100, and 1% bovine serum albumin (BSA) in PBS.

Incubate the sections for 24h 0m 0s at Room temperature in a solution containing the primary antibodies in 1% normal serum, 0.3% Triton X-100, and 1% BSA in PBS.

Next day, thoroughly rinse the sections in PBS.

Incubate the sections in a PBS solution containing the appropriate biotinylated secondary antibody (1:200; Vector Labs, Burlingame, CA, USA) combined with 1% normal serum, 0.3% Triton X-100, and 1% BSA for 1h 30m 0s at Room temperature.

Wash the sections in PBS.

Incubate the sections in an avidin-biotin-peroxidase complex (ABC; 1:100; Vector Labs, Burlingame, CA, USA) solution for 1h 30m 0s at Room temperature.

Rinse the sections in PBS twice followed by a third rinse in TRIS buffer (0.05Molarity (M); 7.6).

Incubate the sections in a solution containing 0.025% 3,3ʹ-diaminobenzidine tetrahydrochloride, 10millimolar (mM) imidazole, and 0.005% hydrogen peroxide in Tris buffer for 0h 10m 0s at Room temperature.

Rinse the sections with PBS.

Mount sections onto gelatin-coated slides, and coverslip with Permount.

Digitalize the slides with an Aperio ScanScope CS system (Aperio Technologies).