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.

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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 undefinedFerricyanide (may keep small frozen aliquots- avoid repeated freeze/thaw).
• 45uL of 210 mg/mL undefinedferrocyanide (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