[Histonet] Re: Histonet Digest, Vol 26, Issue 20

From:"John A. Kiernan"

If your antigen is damaged by fixatives that
contain formaldehyde, and you're not doing
electron microscopy, why not try a simple
coagulant fixative such as Clarke's or Carnoy's
fluid or Puchtler's methacarn? These are good for
conventional carbohydrate histochemistry (except
glycolipids, of course). Check first that your
cell culture plasticware is not soluble in the
fixative. For a thin layer of cells methanol alone
may be OK - as when fixing a blood film.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
"Pixley, Sarah (pixleysk)" wrote:
> Dear John Kiernan and histonetters:
> I read with great interest the article you suggested on the PLP
> fixative. And I searched PubMed for similar articles. But I do not see a
> good analysis of what might be the alternative to PLP. The authors of
> the paper, Hixson et al., suggest that paraformaldehyde and lysine might
> be just as effective as PLP, with lowered cell toxicity, but they don't
> do an analysis of the effects on morphology or antigenicity. Have you
> tried anything like this? Does a combo of PF and lysine provide a better
> fixation of carbohydrate antigens than PLP?  Do you know if these
> authors or anyone else followed up on this and developed and described a
> better fixative in more detail?
> The reason that I ask is that I have an antigen that is particularly
> sensitive to fixation and the monoclonal antibody to it works only with
> the PLP fixative. Supposedly the antigen is the carbohydrate portion of
> a cell surface receptor. But the fixation is so light that I lose almost
> all my cultured cells during processing for IHC (I have very lightly
> attached cultured cells). I have been searching for an alternative to
> PLP for years! So I thank you greatly for this article. I guess my next
> step would be to try different concentrations of paraformaldehyde and
> lysine. But if this has been tried before, I would appreciate any
> suggestions!
> Sarah Pixley

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