LacZ staining of cells
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The bacterial enzyme β-galactosidase (gene LacZ) is frequently used as reporter gene. It can be easily located with a LacZ stain using the artificial substrate X-gal, which turns blue when it is cleaved by β-galactosidase.
This protocol describes the staining of cultured cells containing active LacZ genes.
material
- X-gal stock is 200 mg/ml in dimethylformamide (DMF)
- K ferri-cyanide ( 50mM ) =Fe3- is 0,33 g / 20 ml PBS ( Sigma Cat.# P-8131)
- K ferro-cyanide ( 50mM ) =Fe4- 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 ) | 1 ml | 1,5 ml | 3 ml | 5 ml |
| K ferro-cyanide ( 50mM ) | 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
- make fresh
| 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
sample stains
-
weak LacZ stain from modified mouse embryonic stem cells, 200x
-
strong LacZ stain from modified mouse embryonic stem cells, 100x
