Embryos were fixed in 4% PFA/PBS in PBS O/N at 4 °C. WISH was performed as previously described (Batchelor, 2000 (link)), using plasmids for Brachyury and Sox3 (gift from Dr. Raymond B. Runyan), and Vg1. All WISH were carried out with paralleled control embryos for managing the color development time. For subsequent IHC, embryos were fixed in 4:1 methanol/DMSO at 4 °C O/N.
The next day, embryos were incubated with 4:1:1 methanol/DMSO/30% Hydrogen peroxide at RT and incubated for 2 hr. Then embryos were washed in Tris-Buffered Saline (pH7.6) with 0.1% Tween 20 (TBST) 3 X for 10 min each, and incubated with blocking solution (1% sheep serum in TBST) for 1 hr at RT. Primary antibodies, anti-GFP antibody-HRP conjugated (1:200, Abcam, ab6663), were applied for at 4 °C O/N incubation. Embryos were then washed in TBST 5 X for 1 hr each at RT, followed by 10 min with 0.05 M Tris-HCl (pH 7.6), and incubated for 30 min with DAB (3,3′-Diaminobenzidine)/Tris-HCl (pH7.6) 0.0003% Hydrogen peroxide in the dark. After the reaction was stopped, the embryos were washed in PBS, and placed in 4% PFA at 4 °C for long term storage.
The next day, embryos were incubated with 4:1:1 methanol/DMSO/30% Hydrogen peroxide at RT and incubated for 2 hr. Then embryos were washed in Tris-Buffered Saline (pH7.6) with 0.1% Tween 20 (TBST) 3 X for 10 min each, and incubated with blocking solution (1% sheep serum in TBST) for 1 hr at RT. Primary antibodies, anti-GFP antibody-HRP conjugated (1:200, Abcam, ab6663), were applied for at 4 °C O/N incubation. Embryos were then washed in TBST 5 X for 1 hr each at RT, followed by 10 min with 0.05 M Tris-HCl (pH 7.6), and incubated for 30 min with DAB (3,3′-Diaminobenzidine)/Tris-HCl (pH7.6) 0.0003% Hydrogen peroxide in the dark. After the reaction was stopped, the embryos were washed in PBS, and placed in 4% PFA at 4 °C for long term storage.