material
- X-Gal stock is 200 mg/ml in dimethylformamide (DMF)
- K ferri-cyanide ( 50mM )=K3 is 0,33 g / 20 ml PBS ( Sigma Cat.# P-8131)
- K ferro-cyanide ( 50mM )=K4 is 0,42 g / 20 ml PBS ( Sigma Cat.# P-9387)
staining solution
- make fresh
- heat mix to 50°C before adding X-Gal, in order to avoid precipitation
|
10 ml |
15 ml |
30 ml |
50 ml
|
X-gal ( 200 mg/ml) |
50 µl |
75 µl |
150 µl |
250 µl
|
MgCl2 (1M) |
20 µl |
30 µl |
60 µl |
100 µl
|
K ferri-cyanide ( 50mM )=K3 |
1 ml |
1,5 ml |
3 ml |
5 ml
|
K ferro-cyanide ( 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;
- X-Gal 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 cm-dish
3 ml for 6 cm-dish
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
see also