Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Note: The answer to
Question 2 discusses the suitability of alcoholic and other fixatives
Any thoughts on the shelf life/keeping qualities of Carnoy's fixative?
I always make Carnoy's fixative fresh just before use. Otherwise you will find that the fixing properties will vary if the solution is kept for any length of time. Making up a fresh solution really only takes a few minutes unless you are talking about Lebrun's modification in which the solution is saturated
with mercuric chloride.
Are alcoholic fixatives suitable for immunohistochemistry?
Fixatives containing ethanol are generally not all that great for IHC. About 4-5 years ago we experimented with several fixatives in an attempt to find one that would give us the cellular morphology that we were used to and also be optimal for IHC/ICC. We tested out the following fixatives:
methacarn (Carnoy's with methanol instead of ethanol)
zinc formalin, unbuffered
buffered zinc formalin
The 10% NBF of course gave us the morphology we were used to, and if fixation times were kept to 24-48 hours, epitope retrieval was not required for most antibodies. If tissues needed to be stored longer than 48 hours, they were stored in 70% EtOH until ready to be processed. Of all the fixatives we tested, the worst for IHC was 70% EtOH, then Carnoy's. The best for IHC was 70% MeOH. Cellular morphology for both of these was not all that great. Methacarn gave us both good morphology and good IHC. The zinc formalins gave excellent morphology in many organs, and good IHC staining. It should be noted that the zinc formalins have difficulty penetrating the hematopoietic organs as they react more with the RBCs and therefore penetration is much slower. As those are the organs of interest in our laboratory, we use standard NBF.
We have found that if the tissues are trimmed to a thickness of no more than 3 mm prior to immersion in NBF, fixative solutions are changed at 1 and 12 hours, and after 24 hours in fixative are transferred to 70% EtOH, both cellular morphology and IHC staining are excellent.
One of these days when I have some time I'd like to try some of the other fixatives, as well as some of the commercial ones that are out there, just to see what the total comparisons are going to be like. I would also like to note that Bouin's has seemed to work pretty much all right as I have been doing IHC on some Bouin's fixed testes lately without problems.
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina CB#7400
Chapel Hill, NC 27599