BETA-GAL STAINING OF XENOPUS


β-Gal Staining of Xenopus Embryos

Materials

Staining solution is prepared in PBS + 2 mM MgCl2. Keep wrapped in foil in fridge at 4° C.

Material

For 10 mL

For 1 L

20 mM K3Fe(CN)6

0.06 g

6 g

20 mM K4Fe(CN)6·3H2O

0.08 g

8 g

0.01% deoxycholic acid

0.001 g

0.1 g

0.02% NP-40

2 λ

200 λ

Above materials are in PBS + 2 mM MgCl2

Procedure

  1. Fix embryos for 30 minutes to 1 hour (no more!) in MEMFA (while nutating).
  2. Rinse 2 times with PBS + 2 mM MgCl2.
  3. Transfer to staining solution (max. volume).
  4. Transfer to X-gal mix: add 100 λ of the 50 mg/ml stuff (small brown bottle in -20 °C freezer) per 5 ml of embryo sample in staining solution. Use a minimal volume (5 ml is more than enough for 1 scint. vial) – this stuff is expensive. To develop more rapidly and with a deeper purple color, add 5 λ of NBT (NBT stock as for in situs: 75 mg/ml in 70% dimethyl formamide / 30% water) to 10 ml of final developing mix.
  5. Incubate at 37 °C in the dark (wrapped in tin foil) until desirable stain develops (a few hours to 24 hours).
  6. Rinse in PBS, and twice in Methanol. Samples can be stored here.
  7. Clear in Benzyl Benzoate : Benzyl Alcohol (2:1) if desired. BB:BA is very toxic; it is not water mixable (clean glass with ethanol) and melts plastic, so use it in glass only. It will also leech the stain out of embryos (which become brittle) in a while. You should photograph your samples in a watch-glass.

This protocol is from Levin laboratory under Dr. Michael Levin at Tufts University.








PROTOCOL DEX