Whole-mount Xenopus Immunostaining Protocol

Whole Mount Xenopus Immunostaining Protocol

Adapted from Helen Willsey, modified after Peter Walentek and Susanne Bogusch

Reagents:

-1x MEM with 3.7% formaldehyde

-CAS-Block (Invitrogen #00-8120)

-1X PBS

-1X PBT (PBS + 0.1% Triton X-100)

-1X bleach: per ml= 50uL Formamide, 40uL Peroxide, 910uL PBS

Protocol:

1.     Fix tadpoles in 1x MEM with 3.7% formaldehyde 30min-1hr RT. (NO LONGER THAN 1hr)

2.     Optional: bleach tadpoles for 2hr RT under light box.

3.     Permeabilize by washing 3x20’ RT PBT.

4.     Block in 10% CAS-Block in PBT 1hr RT.

5.     Incubate in 1^o antibody diluted in 100% CAS-Block O/N 4^oC.

6.     Wash 3x10’ PBT RT.

7.     Block again in 10% CAS-Block in PBT 30’.

8.     Incubate in 2^o antibody diluted in 100% CAS-Block 2hr RT.

9.     Wash 3x10’ PBT RT.

10.  Optional: Incubate in Phalloidin-647 diluted 1:40 in PBT 30’ RT.

11.  Wash 3x20’ PBS.

12.  Mount with Vectashield + DAPI.

Category