IMMUNOCYTOCHEMISTRY PROTOCOL

Select coverslips previously fixed in 4% paraformaldehyde and subsequently washed and stored in PBS solution.

Permeabilize: 0.2% TX-100 in PBS for 0h 15m 0s at Room temperature .

Wash 3 x 5 min in PBS.

Wash 0h 5m 0s in PBS. (1/3)

Wash 0h 5m 0s in PBS. (2/3)

Wash 0h 5m 0s in PBS. (3/3)

Block: 50% Sniper in PBS for 0h 15m 0s at Room temperature .

Wash 3 x 5 min in PBS.

Wash 0h 5m 0s in PBS. (1/3)

Wash 0h 5m 0s in PBS. (2/3)

Wash 0h 5m 0s in PBS. (3/3)

Primary antibody: mix desired antibody in PBS containing 5% sniper and 0.1% TX-100 overnight at 4°C(gentle rocking).

Wash 3 x 5 min in PBS.

Wash 0h 5m 0s in PBS. (1/3)

Wash 0h 5m 0s in PBS. (2/3)

Wash 0h 5m 0s in PBS. (3/3)

Secondary antibody: mix desired antibody in PBS containing 5% sniper and 0.1% TX-100.

• Can add DAPI in secondary solution or as a separate step following washing of the secondary.

Wash 3 x 5 min in PBS.

Wash 0h 5m 0s in PBS. (1/3)

Wash 0h 5m 0s in PBS. (2/3)

Wash 0h 5m 0s in PBS. (3/3)

Mount coverslips to slide with Vectashield antifade mounting medium.