Immunohistochemistry free-floating rat brain cryosections  

Collect the cryosections needed for a caudo-rostral representation of each brain region (every fifth or sith sections depending on the section's thickness, brain region, and animal species) into a 24-well-plate (3-4 sections per well)

Wash in PB1X 3 times x 5 min (500 ul) at RT

Incubate sections with endogenous peroxidase blocking solution: PB1X + 3% H₂O₂ (500 µ$$l/well) for at RT

Wash in PB1X 2 times x 5 min (500 ul) at RT

Incubate with blocking solution (500 µ$$l/well) : PB1X + 10%NGS (serum from the same species as the host of the secondary antibody) + 0.3% TritonX-100, at RT

Remove the blocking solution and incubate sections with PB1X+ 5%NGS+0.3% TritonX-100+ primary Ab (500 µ$$l/well) for 24/72 h, depending on the Ab, at +4°C

Wash in PB1X 3 times x 5 min (500 ul) at RT

Incubate sections with adequate secondary biotinylated Ab diluted in PB1X+ 0.3% TritonX-100, at RT

Wash n PB1X 3 times x 5 min (500 ul) at RT

Incubate with Extravidin-peroxidase buffer solution diluited in PB1X+0.3% TritonX-100, at RT

Wash n PB1X 3 times x 5 min (500 ul) at RT

Allow each DAB tablet to reach room temperature before use and Dissolve a DAB tablet in 15 mL of Tris-buffered saline, pH 7.6

Add 12 µL of fresh 30% hydrogen peroxide prior to use

Filter the solution through a 0.2 µm filter immediately before use

Add 500 µl/ well of substrate solution in the dark

Remove and clean al material with bleach

Wash 2 times x 5 min in PB1x

Mount sections on SuperFrost plus slides and let it dry overnight

Dehydrate in consecutive steps of ethanol solutions (50%, 70%, 95%, 100%)

Incubate slides in 2 times x 5 min in Xylene

Coverslip slides with mounting medium, remove bubbles if any, and let dry