[Histonet] FW: CD4 CD8 Zinc Tris PE staining

[gayle callis]

Hello Patsy, 

 

You wrote:   Has anyone tried this:  zinc salt fixed mouse tissue PE stained
for CD4/cd8.  I found some papers that look impressive.

 

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I presume you mean the Beckstead zinc TRIS fixative that is formalin free?
It should work with murine or rat tissues fixed in  ZnTRIS  since the
tissues can then be paraffin processed, but avoid any solvent that have NBF
carry over so these antigens are not compromised by that sniiff of aldehyde.
The fixative can be purchased from BD Bioscience/Invitrogen.   However, PE
(phycoerythrin) fades rapidly before your eyes while you are looking at the
stained sections.  Been there, and seen it happen resulting in a lot of
wasted time and effort.    PE is for FACS not fluorescence microscopy.
Not only that, but doing direct immunofluorescence using CD4 or CD8
conjugated to a fluorophore will result in poor to no staining since the
fluorphore molecules in close approximation to each other will exchange
electrons and quench fluorescence.   It is better to do CD4 or CD8 then come
back with an excellent secondary e.g. Jacksons donkey anti mouse F(ab')2
frag of IgG conjugated to Alexa 594  OR use a biotinylated CD4 and CD8 and
come back with Streptavidin Alexa 594.   Double staining using this fixative
should be very possible, even triple IF.    

 

I have several publications for this fixative including the original
Beckstead (for human) and Nitta et al (for murine) CD marker publications.
We tried ZnTris for chromogenic IHC with success but didn't continue to use
it since we did the work much faster with frozen sections.    We found this
fixative did not work well on fresh tissue frozen sections though, and
preferred to use our favorite 75% acetone/25% absolute ethanol for 5 minutes
at RT  on air dried frozen sections then into buffer from this solvent
fixative (sections were not alllowed to air dry again after this fixaton).  

 

As for papers you have seen, can you give the references please?  

 

Gayle Callis

HTL/HT/MT(ASCP)