Mouse CD4 and CD8

From:Gayle Callis

Unfortunately, frozen sections with acetone or acetone/alcohol fixation OR
a nonformalin fixative touted by BD Pharmingen called zinc fixative (DO not
confuse this with zinc formalin), is zinc salts in TRIS buffer can be used
for paraffin sections seem to be the norm for staining CD4 and CD8.  

The CD4 and CD8 formalin fixation/paraffin embedded (FFPE) problems (even
with retrievals, digestion, or amplification) are the reason we do only
frozen sections for these CD markers. I know people insist on paraffin with
good morphology and many dislike doing frozens, but we find cryotechnics
actually faster and with good morphology acetone/alcohol fixation followed
by glucose oxdidase block for endogenous peroxidase, and using the DAKO
DAB+/DAKO DAB enhancer at end of protocol. We have our dilution on a 0.5
mg/ml as high as 1:15,000. Hard to beat these results. 

CD4 and CD8 are not the only markers that can present problems with FFPE on
mouse tissues.  

Aldehyde fixatives just crosslink the antigens too strongly, and why PLP
may work better than 4% paraformaldehyde may be differences in combination
of chemicals with these two fixatives. I would have to reread the Nakane et
al publication again for details.  Although it is reported in the
literature that PLP fixation permits adequate murine CD marker staining,
even CD4 and CD8, we have never liked it very well. I'm not surprised they
have high concentrations of antibody, since PLP has paraformaldehyde.
Problems with FFP/any aldehyde fixative and having to perform multiple
staining on one experimental tissue (CD4, 8, B220, MAC 1, PNAd, MADCAM,
etc, etc) led to our decision to stay with frozen section technics
particularly when doing serial sections, even double staining technics on
adjacent sections and far less work overall.  

I suggest you try the Zinc (TRIS buffer) fixative, the antibody
concentrations are comparable to those with acetone or acetone/alcohol
fixation and works nicely on paraffin embedded tissues, morphology is
reported to be good also. I will be happy to send you the protocol via
private email file attachment. It is also found in Histonet Archives,
discussed recently/many times.  

As for verifying the Whiteland group results, a parallel study could be
done - one using PLP with their methods and materials, and the other with
tried and true frozen section technics with an extensive dilution panel and
I would toss in the ZINC TRIS buffer fixation as a bonus just to see how
this works.  Since frozen sections are the usual way companies test their
antibodies for immunohistochemistry, you know that they work - a baseline
of positive proof staining. I realize this is work for you, but would
answer your questions in house. 

Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

<< Previous Message | Next Message >>