Dear John

We've been using Novocastra's clone 1F6 for several years with reasonable
success (concentration 2.5 ug/ml, corresponding to a dilution of 1:20,
depending on the lot - insist with Novocastra that you want to know the Ig
concentration, they have it for every lot!). We pretreat the slides in the
pressure cooker in 10 mM Citrate buffer, pH 6.0 (1.5 l of buffer, heat to
boil, immerse slides in a metal racket, close lid, reduce heat so that it
takes 3-4 minutes for the pressure to reach 1 atm [15 lbs], continue for 2
more minutes, remove cooker from hot plate, put under cold water, open and
transfer slides immediately to H2O or TBS at room temp) as we don't like
what EDTA does to our sections (inferior morphology). If CD4 doesn't stain,
this usually is a problem with fixation. In fact, we get reasonably good
staining with most of our own sections (tissue fixed for approx. 24 hours in
neutral buffered formalin) and have a lot of problems with material that
comes from research labs, where some enthusiastic researcher may have put
his tissues into formalin of unknown quality - for several days. The antigen
recognized by 1F6 seems to be extremly sensitive to overfixation and other
variants of ill-treatment. Sometimes we had more success when we did 24 hour
incubations for the primary at room temperature (instead of the usual 60
min). Cover your tissue with the antibody and a coverslip, put it in a humid
chamber and leave it on the bench until the next day. It doesn't produce
miracles, but it may help. And first of all: fixation, fixation, fixation.
Good luck!

Andi Kappeler
Immunohistochemistry Lab
Institute of Pathology, University of Bern
3010-Bern, Switzerland