I am looking for a good method to fix cells after primary antibody.
Right now I am using 4% Parformaldehyde.
Any other good ideas?
I've always liked 4% paraformaldehyde. Are you finding that it doesn't work well with what you're trying to do?
You can fix with 70% ethanol chilled and do your fixation for 15-20 min on ice , but you will have to experiment with your solution to avoid excessive shrinkage during fixation. You can also use methanol but fixation takes longer (~1h)
Also you can fix in 1 % glutaraldehyde, incubating the
cells for at least 15-20 min @ RT.
- Stop fixation process by adding 1 ml icecold PBS.
Zamboni fix (PFA and picric acid) is preferred fixative in my lab for ICC, especially when PFA alone does not facilitate desired results. Works exceptionally well in embryonic tissue too.
Please keep in mind that different antigen has its own optimal fixative so onby by trial and error could you know which is the best .Good luck
Thank you all for the advice.
Lately I have started doing my one PFA from Powder. I guess the PFA that we bought (16%) wasnt good.
So now it is working much better.
And, yes thats true depending upon Ab's the fixation could be changed to be more efficient.