Re: cd3, cd4, cd8, and cd68 for mouse tissue

From:Gayle Callis

CD4 aned CD8 will NOT work on FFPE tissues, you have to do frozen sections.
Check Histonet archives. These antigens cannot be retrieved after any
aldehyde fixation.   CD3 does work (in archives) although we do all murine
IHC with frozen sections since we usually have to do a huge panel of
antibodies on one tissue collected and cannot do both frozens and FFPE on
adjacent sections when this is used since all antibodies are correlated
withing 20 um of each other. Acetone alcohol fixation is used, excellent
results, and glucose oxidase endogenous peroxidase blocking. Methanol
peroxide blockers are always avoided for CD markers. 

We do not use kits, do inhouse dilutions using SA-HRP from Biosource, and
van der Loos AEC recipe.  

BD Pharmingen or SEROTEC for primary antibodies, monoclonals is our
preference, rat or hamster antiMouse.   CD3 from DAKO is reported to work
on FFPE tissue, but not sure about CD68, check with vendor, most of the
time BD recommends frozens because they do not test on FFPE.  One can
always the Zinc TRIS buffer fixative (sold by Pharmingen) and avoid antigen
retrieval altogether, using alk phos staining method, dilutions similar to
frozen section work. 

We buy goat anti rat secondaries from TAGOImmunologicals (Biosource)
F(ab')2 fragments of IgG adsorbed to mouse or Jackson's Donkey antiRat
F(ab')2 fragments adsorbed to mouse.  Southern Biotechnology has excellent
anti rat whole IgG secondaries, adsorbed to mouse.

Most of our murine IHC is done using biotinylated primaries to avoid
secondary antibodies altogether, making staining faster, certainly clean.

At 10:36 AM 7/2/2003 -0600, you wrote:
>What vendors antibodies and procedures are being used for ffpe mouse tissue
>for these antibodies.
>Thank you,
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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