Immunohistochemistry on free-floating cryosections

Collect the cryosections needed for a caudo-rostral representation of each brain region (every fourth section or every sixth section dependeing on the section thinknes, brain region and animal species) into 24-well-plate (3-4 sections per well).

Wash 3x5 min in TBS 1X. Put 500ul per well and aspirate the liquid with an air-pump equipment.

Incubate sections in endogenous peroxidase blocking solution: TBS 1x + 3% H2O2 + 10% methanol for 10 min (500uL/well)

Wash 3x5 min in TBS 1X.

Incubate sections in blocking in TBS 1X + 5% NGS or NDS (500uL/well) 1h at RT

Incubate sections inTBS 1X + 2% NGS or NDS + primary Ab 24/72h (it depends of the Ab) at 4ºC (cold room).

Wash 3x5min in TBS 1X.

Incubate sections in 2% NGS or NDS + Secondary Ab 1h at RT .

At this step it is important to prepare ABC solution and let it, at least, 30 min on the shaker

Wash 3x5min in TBS 1X.

Incubate 1 hour at RT with ABC solution (Ultra-Sensitive or Standard ABC Peroxidase Standard Staining Kit).

Wash 3x5min in TBS 1X.

In aluminium foil: DAB Standard Kit (1 drop of reagent B in 1 mL of reagent A, gives rise to brown staining), or Vector SG (3 drops Chromogen + 3 drops Hydrogen Peroxide in 5mL PBS, gives rise to blue staining).

Put 500 uL on each well and put a cardboard box on it to keep darkness for a time ranging from 3-15 minutes depending on the antibody used.

Remove with an air-pump equipment and clean the material with bleach.

Wash 3x5min in TBS 1X.

Mount sections into slides and let it dry overnight.

Incubate slides in consecutive ethanol solutions (1 min in ethanol 70%-1 min in ethanol 95%-1 min in ethanol 100%).

Incubate slides in 2x5 min in Xylene.

Put a line of mounting medium (DPX) by slide. Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles.

Let dry the slides in the hood overnight.