Whole Mount EB Immunostaining

Materials:

4% formaldehyde

PBS

2% FBS diluted in PBS

0.1% Triton-X

Primary antibody

Secondary antibody

DAPI

Procedure:

1) Wash EBs at room temp twice with PBS

2) Fix EBs in 4% formaldehyde at room temperature overnight

3) Wash EBs in new 2% FBS diluted in PBS every hour for 5 hours

4) Permeabilize with 0.1% Triton-X for 1 hour

5) Wash EBs with PBS for 1 hour

6) Stain with primary antibody overnight

7) Wash 5 times with PBS over an hour

8) Stain with secondary antibody overnight

9) Wash 5 times with PBS over an hour

10) Stain with DAPI overnight

11) Wash with PBS and visualize