Re: [Histonet] Murine leukocytes on paraffin?

From:"Gayle Callis"

CD4 and CD8 will never work on Paraformaldehyde fixed, paraffin embedded 
murinE tissue no matter what retrieval OR enzyme digestion you try.  Sad, 
but true!

Macrophage antibody, F4/80 will work with retrieval (in histonet archives 
several times) and I am not sure about B220.   We only do frozen sections on 
fresh snap frozen tissues, fixed with 25% absolute ethanol/75% acetone, a RT 
fixation on thoroughly air dried frozen sections (overnight).  If you want 
to stain adjacent sections for CD4, CD8, F4/80, and B220 - then you would 
have to do cryotomy.  Only one of these four antibodies will work on FFPE or 
PFAF-PE murine tissue.

Serotec has a good range of murine monoclonals, but we purchase most of our 
murine antibodies from eBioscience or Invitrogen (BD Pharmingen).

There is a non-aldehyde fixative you can purchase from BD Pharmingen i.e. 
Invitrogen that they call zinc fixative. It is FREE of  formalin and 
paraformaldehyde, you can make it up yourself.  We refer to this formalin 
free fixative as Zinc TRIS.   You can go J Histochemistry and Cytochemistry, 
for a publication by Jay Beckstead.   He developed this fixative for human 
lymphoma CD marker staining and it was then published for use with rodent 
tissue H. Nitta et al, in a now defunct journal called  Cell Vision. 
Whatever you do, do NOT confuse Zinc TRIS buffer as zinc formalin as the 
latter will not work.

This fixative was recently discussed on Histonet (last month).  If you go to 
the archives you can probably pick up the recipe and further discussion.

Gayle M. Callis

----- Original Message ----- 
From: "Mikael Niku" 
Sent: Friday, August 15, 2008 1:13 AM
Subject: [Histonet] Murine leukocytes on paraffin?

> Dear Histonetters,
> I'm looking for antibodies to identify basic leukocyte types on murine
> PFA-fixed paraffin sections (such as tissue macrophages, B cells, and
> CD4+CD8).
> I just noticed that AbD Serotec seems to offer a fairly nice range of rat
> and hamster monoclonals for these purposes. Any experience on these
> products, do they really work well on paraffin?
> With best regards,
> Mikael
> -----------------------------------------------------
> Mikael Niku, PhD, university lecturer
> University of Helsinki, Finland
> URL:
> - What do I think of western civilization?
>   I think it would be a good idea!
>                                            Gandhi
> -----------------------------------------------------
> _______________________________________________
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