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.
Start => Dechorinated zebrafish embryos.
- Add embryos to siliconized microcentrifuge tubes (no more than 100 per tube).
- Remove excess H2O.
- 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.)
- Rinse embryos 3x in PBST for 5-10 minutes.
- Add 0.5mL of freshly made staining solution until blue color develops (@RT - 37 degrees).
- Rinse embryos 2x in PBST for 5- 10 minutes.
- Rinse embryos 2x in Methanol for 5-10 minutes to dehydrate prior to clearing.
- Clear embryos (if desirable) in 2:1 :: benzyl benzoate:benzyl alcohol.
- 0.8% NaCl
- 0.02% KCl
- 0.02M PO4
- pH 7.3
- 8 g NaCl
- 0.2 g KCl
- 16 mL 1M Na2HPO4
- 4 mL 1M NaH2PO4
- 980 mL H2O
- PBS + 0.1% Tween 20
For 500 mLs PBST
- Add 500 uL Tween 20 to 500 mLs PBS
- 900 uL of Spermidine solution
- 100uL of 2% Xgal in DMF
- 50uL of 165 mg/mL K*Ferricyanide (may keep small frozen aliquots- avoid repeated freeze/thaw).
- 45uL of 210 mg/mL K*ferrocyanide (may keep small frozen aliquots- avoid repeated freeze/thaw).
pH should be between 7.0 and 7.5
Spermidine Solution (for 10mL stock)
- 40 mg spermidine (Final concentration 4mg/mL)
- 20 uL 1M MgCl2 (Final concentration 2mM)
- 10 mL PBST