Immunofluorescence free-floating rat brain cryosections

Collect the cryosections needed for a caudo-rostral representation of each brain region (every fifth or sixth section 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 with blocking solution (500 µ$$l/well) : PB1X + 10%NDS (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%NdS+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 fluorescent Ab diluted in PB1X+ 0.3% TritonX-100, at RT

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

Incubate with DAPI diluted in PB1X, to stain nuclei

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

Mount sections on slides and let it dry

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