Whole Mount Immunohistochemistry

Throughout the entire protocol, the embryos should be gently agitated to improve penetration of the tissue
At E10-11, pinch hole in telencephelon and at base of head so that neural tube will flush easily. This will help prevent Ab trapping.

Method

Fix embryos in 4% paraformaldehyde/PBS at 4°C O/N.
Bleach embryos in 5:1 H₂O₂:PBS at RT for 3-5 hours.

You can then run through methanol series and store in 100% methanol at -20°C if desired. You will then need to rehydrate to PBS before proceeding with protocol.
Incubate 2X in PBSMT at RT for 1 hour each.
Incubate at 4°C O/N with primary antibody in PBMST. A 1:100-250 dilution is generally used.
Wash 2X in PBMST at 4°C, and 3X in PBMST at RT for 1 hour.

Note: Increase volume and number of changes if possible.
Incubate 4°C O/N with secondary antibody diluted in PBSMT. Peroxidase-coupled secondary generally used at a 1:200-500 dilution.
Repeat wash steps a second day, adding a final wash in PBT for 20 minutes.
Incubate embryos in 0.3 mg/ml DAB and 0.5% NiCl₂ in PBT at RT for at least 20 minutes.

NiCl₂ may precipitate a little, so add extra water.
Add 0.00003% H₂O₂ (yes, really) and incubate at 4°C O/N.
Rinse in PBT for 2 days or longer.
You can dehydrate and store in 100% methanol.
Alternatively, embryos can be cleared in benzyl alcohol:benzyl benzoate (1:2 BABB).

Note: Use glass containers with BABB!!