Immunohistochemistry of free floating slices

Fix with paraformaldehyde (PFA) 4% at Room temperature.

• 4g of PFA

• Dissolve in 100mL de PBS (pH 7.3) previously heated to around 60°C.

• Mix the solution until it completely clears up.

• Filter and store at 4°C for a duration of 336h 0m 0s.

Rinse with PBS 1X (3X 10 min) under agitation.

Rinse with PBS 1X for 0h 10m 0s under agitation (1/3).

Rinse with PBS 1X for 0h 10m 0s under agitation (2/3).

Rinse with PBS 1X for 0h 10m 0s under agitation (3/3).

Permeabilize and block the non-specific primary antibodies bindings in a 24-well multiwell plate

by incubating under agitation for1h 0m 0s with0.5mL to1mL of a BSA enriched ab solution 10mL of ab solution for1g of BSA).

All the subsequent steps are done using the following ab solution:

• 95mL of PBS 7.3

• 20mg of NaN3 (final 0.02 %)

• 300µL of Triton X-100 (final 0.3 %)

• 500mg of BSA (final 0.5 %)

• 5mL of goat serum (final 5 %)

Dilute the primary antibodies in the ab solution and apply 0.5mL per well (up to 10 slices)

1h 0m 0s at Room temperature under agitation.

Rinse the slices with PBS (3X 10 min) under agitation.

Rinse the slices with PBS for 0h 10m 0s under agitation (1/3).

Rinse the slices with PBS for 0h 10m 0s under agitation (2/3).

Rinse the slices with PBS for 0h 10m 0s under agitation (3/3).

Dilute the secondary antibodies in the ab solution and apply 0.5mL per well (up to 10 slices) for 2h 0m 0s at Room temperature under agitation.

Rinse the slices with PBS (3X 10 min) under agitation.

Rinse the slices with PBS for 0h 10m 0s under agitation (1/3).

Rinse the slices with PBS for 0h 10m 0s under agitation (2/3).

Rinse the slices with PBS for 0h 10m 0s under agitation (3/3).

Mount the slices on charged (+) glass slides submerged in PBS.

Let the mounted slices dry (about 0h 10m 0s).

Seal the slides with 100µL-120µL/slide of Fluoromount G and a rectangular glass coverslip and seal the coverslip to the slide with nail polish.