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Bgal Staining- Whole Mount

Bgal Staining- Whole Mount


Objective:

Bgal staining allows identification of embryonic tissues/cells expressing lacZ marker protein by development of pigmented (blue) product in the presence of lacZ enzymatic activity.

Procedures:

    Start => Dechorinated zebrafish embryos.
  1. Add embryos to siliconized microcentrifuge tubes (no more than 100 per tube).
  2. Remove excess H2O.
  3. Gently fix by adding ice cold 4% paraformaldehyde in PBS and incubating on ice for ~30 minutes. (Fixing time is dependent on embryo age- older embryos (>24 Hrs) may require more time.)
  4. Rinse embryos 3x in PBST for 5-10 minutes.
  5. Add 0.5mL of freshly made staining solution until blue color develops (@RT - 37 degrees).
  6. Rinse embryos 2x in PBST for 5- 10 minutes.
  7. Rinse embryos 2x in Methanol for 5-10 minutes to dehydrate prior to clearing.
  8. Clear embryos (if desirable) in 2:1 :: benzyl benzoate:benzyl alcohol.

Reagents/Solutions


This file was created on **** by Karl J Clark (kclark@biosci.cbs.umn.edu).

Last modified July 12, 2000