Cell Fixation - Ethanol or Methanol? - (Sep/14/2006 )

Hi

I've seen some people use either -20oC ethanol or methanol for cell fixation for imaging. Just wondering if there is any particular difference in using either?

I'm currently using -20oC methanol for fixation.

Hi!

If you only stain cells, I guess there is no difference.

I usually use Methanol for fixation, but I was told that if you stain some filters, e.g. PCF filters Ethanol is recommended. Why it is better to use Ethanol then I don't know... Maybe there is interaction between the surface of the filter and the Methanol...?

In other cases it doesn't matter (until now ).

some fixations methods might preserve the cell morphology better. So one has to try different ones if one is not satisfied with a particular fixative.

"There are always many ways to skin a cat"

YOU SHOULD ALWAYS OPTIMISE YOUR CONDITIONS


I look after a Leica SP1 Confocal and have end users coming to me with cells that they have stained. I ask every user if they have optimised their conditions and they look blankly at me.

The fixation protocols could include :-

1,2,3,4 % Ethanol for 5,10,20,30,60 minutes
1,2,3,4 % Methanol for 5,10,20,30,60 minutes
1,2,3,4 % Glutaraldehyde for the same times as above
1,2,3,4 % Paraformaldehyde for the above times


And there are other fixatives, blocking steps and other variables such as primary and secondary antibodies.

I have tried everything, but recently went with a 4% paraformaldehyde-0.25% Triton X-100 for 15 mins at room temp. We do a lot of immunoflouresence in our lab and this has produced the best results. Good luck.

If it were easy, everyone would be doing it.