Protocol
Materials
BUFFERS:
Sodium Phosphate Buffer (.5M stock solution) pH 8.0 (2L)
141g Na2HPO4 (anhydrous; 268.07g for heptahydrate)
8 ml 85% phosphoric acid
qs to 2L
FIX (can prepare ahead except for gluteraldehyde) makes 50 ml total
0.2 ml 50% gluteraldehyde
2.5 ml 100mM EDTA (pH 8.0)
0.1 ml 1M MgC12
47 ml 0.1 M phosphate buffer
WASH
0.3 ml 1M MgCl2
2ml 1% deoxycholate
2ml 2% NP40
195.6 ml 0.1 M phosphate buffer
STAIN (make ahead except for x-gal -store in dark [foil-covered tube]; after x-gal added, stored 4°C; can be re-used)
2 ml 25 mg/ml x-gal in dimethylformamide (stock, store -70°C)
0.106 g potassium ferrocyanide (Sigma P9387)
0.082 g potassium ferricyanide (Sigma P8131)
48 ml wash buffer
Methods
Procedure
1. dissect embryos in PBS; wash at room temp in 0.1 M phosphate buffer
2. fix for 15 to 20 minutes at room temp with mixing
3. wash (3) x 15 minutes room temp in wash buffer
4. place in stain at 37°C; stain starts to come up in about one hour, but if negative embryos do not have any background staining*, it is safe to leave overnight
Troubleshooting
* If there is some specific background, higher pH of buffer helps reduce background
d10 embryos -yolk sac stain;
d12 embryos -thin stripe of staining in hindbrain
