mouse CD4 and CD8 on paraffin
Dear collegues,
I know that the question of staining of CD4/CD8 in paraffin-embedded
mouse tissues has been asked several times during the last years. I am
desperately looking for a CD8 antibody which works in mice. Until now we
have tried antibodies from Serotec (KT15), BD Pharmingen (53-6.7) and
Santa Cruz (sc7979, clone D-9) and none of them worked. Regarding the SC
antibody: this one stained macrophages and dendritic cells but certainly
not CD8 lymphocytes, although the datasheet implied that it should work
on paraffin. We have tried this with several pre-treatments
including heating with microwave or steaming device (1 hr.), trypsin,
protease and in addition tyramid-biotin enhancement. Recently the work
from Whitefield et al., J Histochem Cytochem; 43: 313-320 (1995) came to
my attention and they describe staining for CD4 and CD8 on
periodate-lysine-paraformaldehyde fixed, paraffin-embedded material. The
pictures they show are very convincing but I still have soem doubt. I
know and used this fixation technique and can understand that this might
increase the number of epitopes free for staining with the antibodies. It
however is hard to believe that there are so much more epitopes that it
results in a staining which is not possible with 4% paraformaldehyde and
intensive enhancement with the biotin-tyramid system (with this we
generally can dilude antibodies 10 times as high). So, coming to my
question: Is there someone who has been able to verify this staining
of CD4 and CD8 in mouse material? The Whiteland group uses incredibly
low dilutions for some antibodies (undiluted for KT15, W3/25, 1:5
for OX42 and 1:20 for CD4 (RM4-5). Can this make the
difference?
Thanks in advance for your commentaries!
Jan
-----------------------------------------------------
Jan Bauer, Ph.D
Div. of Neuroimmunology
Brain Research Institute
Spitalgasse 4
A-1090 Vienna
Austria
tel: +43-1-4277 62813
fax: +43-1-4277 9628
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