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