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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

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