Dear All,

Many weeks ago I asked if anyone had used Novocastra CD4 mouse
monoclonal antibody on paraffin sections  and with what
results.  I promised to get back with a summary of suggestions and
subsequent results.  Sorry for the long delay - it isn't a reflection
on how long it's taken to put the ideas into practice! Too much other
stuff to do - the joys of working on a PhD :-).

Anyway, thanks for all the responses, and to the good people at
Novocastra/Vector who were incredibly helpful too.

There appears to be many different methods of antigen retrieval in
use with this antibody, with the common factor of a retrieval time of
15 minutes or longer (up to 40 minutes in some cases).  I've found
that either 15  minutes of microwave antigen retrieval in 1mM EDTA
buffer pH8, or 5 minutes in a microwavable pressure cooker using the
same buffer is optimal.  A crucial point is that the buffer must be
pre-boiled (vigourously!) before immersing the slides.  Ensure that
the slides are cooled in the buffer for aprox. 20 minutes by standing
the container in cold running water.

An essential point is that pH is critical.  TBS must be pH7.6.  Check
each time it's made up, if it's usually made up fresh from a 10x
stock.   Additionally, I found that changing the antibody diluent
from 20% normal rabbit serum in TBS (which we usually use as
standard) to 10% in TBS also helps.  Apparently, a high concentration
of serum in the ab diluent can alter the pH from the optimal pH of
7.6.

Once again, thanks for all the suggestions.  The HistoNet is a
great source of information - particularly the recent discussion
concerning negative controls for immunohistochemistry.

Best Wishes,

Liz Sabin.

Liz Sabin
Department of Pathology
University of Edinburgh
Medical School
Teviot Place
Edinburgh
EH8 9AG

email. esabin@srv4.med.ed.ac.uk