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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

  1. Fix embryos in 4% paraformaldehyde/PBS at 4°C O/N.
  2. 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.

  3. Incubate 2X in PBSMT at RT for 1 hour each.
  4. Incubate at 4°C O/N with primary antibody in PBMST. A 1:100-250 dilution is generally used.
  5. 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.

  6. Incubate 4°C O/N with secondary antibody diluted in PBSMT. Peroxidase-coupled secondary generally used at a 1:200-500 dilution.
  7. Repeat wash steps a second day, adding a final wash in PBT for 20 minutes.
  8. 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.

  9. Add 0.00003% H2O2 (yes, really) and incubate at 4°C O/N.
  10. Rinse in PBT for 2 days or longer.
  11. You can dehydrate and store in 100% methanol.
  12. Alternatively, embryos can be cleared in benzyl alcohol:benzyl benzoate (1:2 BABB).

    Note: Use glass containers with BABB!!

Solutions

PBSMT

  • 2% Milk
  • 0.1% Triton X-100 in PBS

PBT

  • 0.2% BSA (Sigma A-4378)
  • 0.1% Triton X-100 in PBS
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DAB

  • Diaminobenzidine [Carcinogenic]

© 2014 The University of Texas MD Anderson Cancer Center