Hello all!
I have som trouble staining beta galactose in my MC3T3 cells.
Here is my protocol:
- Cells stressed with H202 treatment.
- washed twice in PBS
- fixated in 4% paraformaldehyde 5min at 4 degrees.
- washed 3times ×3min in PBS
- staining solution added (freshly made. Containing 1mg/ml X-gal, 150mM NaCl, 40mM sodium phosphate and Citric acid, 5mM potassium ferrocyanide and potassium ferricyanide, 2mM MgCl2, pH 6)
-incubate over night, 37degrees in a non CO2 chamber
What is missing or could be optimised for the staining to work?
Grateful for any help!
Hi Elina!
I didn´t do it in cell culture but in snap frozen brain tissue; I was using PFA at 16% during 15 minutes (just for you to know) I would suggest long crosslink time.
But I will recomend for sure is the B-galactosidades reporter gene staining kit from Sigma. Used it a lot. worked very well.
Thanks for your answer! I was under the impression that If the sample Is fixated for too long it might deactivate the enzyme thus the staining would not work. But it works for you at 15min?
Yes,
you can try with 5-10-15 mins to find your optimal crosslink time.
Maybe 15 is ok for tissue but I think 10 minutes will work for you.
keep me updated!
Hi Oscar!
Thanks again for the input. When i fixated the cells in 2% formaldehyde and 0.2 % glutaraldehyde for 5min the staining seemed to work, although with some background but still!
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