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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.

-xtrios-

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 wink.gif ).

-Chakchel-

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.

-scolix-

"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.

-Rhombus-

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.

-Dr.C-