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. 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.

Reagents/Solutions PBS

• 0.8 NaCl
• 0.02 KCl
• 0.02M PO4
• pH 7.3 For 1L
  • 8 g NaCl
  • 0.2 g KCl
  • 16 mL 1M Na2HPO4
  • 4 mL 1M NaH2PO4
  • 980 mL H2O
  PBST
• PBS + 0.1 Tween 20 For 500 mLs PBST
  • Add 500 uL Tween 20 to 500 mLs PBS
  Staining Solution (for 1 mL)
• 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