Lac Z staining mouse embryos


Posted by cpark
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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