material
 XGal stock is 200 mg/ml in dimethylformamide (DMF)
 K ferricyanide ( 50mM )=K3 is 0,33 g / 20 ml PBS ( Sigma Cat.# P8131)
 K ferrocyanide ( 50mM )=K4 is 0,42 g / 20 ml PBS ( Sigma Cat.# P9387)
staining solution
 make fresh
 heat mix to 50°C before adding XGal, in order to avoid precipitation

10 ml 
15 ml 
30 ml 
50 ml

Xgal ( 200 mg/ml) 
50 µl 
75 µl 
150 µl 
250 µl

MgCl_{2} (1M) 
20 µl 
30 µl 
60 µl 
100 µl

K ferricyanide ( 50mM )=K3 
1 ml 
1,5 ml 
3 ml 
5 ml

K ferrocyanide ( 50mM )=K4 
1 ml 
1,5 ml 
3 ml 
5 ml

PBS ( up to...) 
7,9 ml 
8,5 ml 
23,5 ml 
39,6 ml

fixation solution

10 ml 
15 ml 
30 ml 
50 ml

formaldehyde/methanal ( 37 %) 
541 µl 
811,5 µl 
1,62 ml 
2,7 ml

glutaraldehyde ( 25 %) 
40 µl 
60 µl 
120 µl 
200 µl

PBS (up to...) 
9,42 ml 
14,13 ml 
28,26 ml 
47,1 ml

common mistakes / tips
 time of fixation is crucial; overfixing cells will reduce LacZ activity
 some cell types are easy to detach by squirting liquid onto the dish; be careful;
 XGal will precipitate if solution cools too much
steps
 carefully wash cells once with PBS (avoid detaching cells by strong pipetting)
(volume is around 5 ml for 10 cmdish
3 ml for 6 cmdish
500µl for a 24 well)
 add fixative solution
 incubate 2 min (Time is important !)
 carefully wash 3 times with PBS (1st wash must be quick to avoid overfixation in the last samples to be washed)
 add staining solution
 incubate over night at 37°C