LacZ staining of whole mouse embryos

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The bacterial enzyme β-galactosidase (gene LacZ) is frequently used as reporter gene in mouse biology. Its expression can be easily located with a LacZ stain using the artificial substrate X-gal. In the case of a LacZ construct under an endogenous promoter, this can reveal expression patterns during embryo development.

material

product price estimates from 2007

0.2% glutaraldehyde fixative

  • 0.4ml 25% glutaraldehyde (GA); store at -20°C
  • 0.5ml 0.5M EGTA (e.g. Sigma, 3889-100g, 230€, stable, MW 380 g/mol)
  • 5ml 1M MgCl2 (MgCl2·6H2O 203g/mol)
  • 44ml PBS

LacZ wash buffer

for 500ml LacZ wash buffer:

  • 1ml 1M MgCl2
  • 5ml 1% sodium deoxycholate (NaDC) Sigma 6750; dissolve in H2O store in fridge
  • 5ml 2% NP40, dilute in H2O (e.g. Roche, 11332473001, 5x10ml 10%, ~80€)
  • 489ml PBS

LacZ staining buffer

stock solution; store -20°C

  • 1g X-gal (5-bromo-4-chloro-3-indoxy-β-D-galactopyranoside, CAS 7240-90-6, Carl Roth 1g 72€ #2315, Sigma 16665-1G 173€)
  • 40ml DMF

for 100ml staining buffer

  • 4ml stock
  • 96ml LacZ wash buffer
  • 0.21g (5mM) K-ferrocyanide (e.g. Sigma, P9387-100g, ~10€; shelf life ~2y; light sensitive)
  • 0.16g (5mM) K-ferricyanide (e.g. Sigma, P8131-100g, ~10€; shelf life ~3y; light sensitive)

steps

  • transfer the embryo into 1.5ml tubes
  • fix the embryo at 4°C for 30-60min
  • wash the embryo 3x 20min at RT to remove fixative and permeabilise
  • stain embryos in the dark with LacZ staining solution
  • wash embryos in PBS, store in PBS in fridge for some days to intensify stain
  • store in 70% EtOH indefinitely

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