Hello Nancy,

Actually, the best fixative for immunohistochemistry is the one that preserves the epitope in the tissue of interest.  In many cases, that is 4% paraformaldehyde in Sorensen's Phosphate Buffer with a pH between 7.2 and 7.4.  However, it is not the only fixative that people use, so I would search the literature for your particular antibody to see what was successful. Of the fixatives you have tried, neutral buffered formalin may have too much methanol in it for immunohistochemistry (but you never know until you try), and formol alcohol may have too much alcohol, which may denature the epitope. 

The manner in which you embed your tissue also has an effect on successful immunohistochemistry.  I prefer to cut cryosections so that I don't have to use the epitope recovery methods necessary in paraffin-embedded tissue.  However, there are plenty of labs who do it, and you can then you still use some sections for H&E.

I personally prefer to fix tissues with 4% paraformaldehyde (or other percentages) by transcardial perfusion of the animal, rather than by immersion fixation.  The benefit of formal alcohol is that is fixes very rapidly, so you don't have the problem of unfixed tissue in the center of the organ.  Paraformaldehyde can only penetrate at a certain speed, and so immersion fixation can take a long time unless you cut the tissue up into small pieces.  But there are people who successfully immersion fix tissue, in some cases by placing it at 4 C while fixing overnight.

Good Luck!

Jill

10% Formalin on phosphate buffer ( Lilly`s Formalin), the volume ratio of tissue and fixative solution 1:20-25.

We stain spleen cryostections after PFA 4% overnight fixation by immersion at 4ºC. If you need to leave the tissues fixing a longer period of time, then we normally do 2% PFA at 4ºC (over-weekend for instance).

10% Formalin on phosphate buffer.

We use 4% PFA as a fixative and it usually works.

Thanks

neutral buffer formalin 10% pH 7.2-7.4 it works good