Summary: immunocytochemistry

Hello all, here is a summary of the responses to my original posting
(23/03/04):

'Hello Flow-ers, this is a basic question...I would like to
double-indirectly label intracellular antigens with fluorescent
secondary reporters on cells grown on chamber slides. What I would
really like is some helpful basic practical guides as to techniques,
especially hints and tips for avoiding pitfalls as we all know the
devil
is in the detail. Although I have some flow cytometry experience, I've
found many of the references reporting ICC results often state that
methods were...'as previously described', but the same message may pop
up in the paper referred to, or ....'performed using routine methods'.
Not very helpful. Recommendation of a good website or even reference
book would appreciated. I will post a summary of all responses to the
group in a couple of weeks.'

There were only a few responses but mainly recommending a number of
manufacturers websites, one which I found particularly useful was

'Hi Dr Rizzo,
Have you  checked out vector labs website at
www.vectorlabs.com. When I was doing
immunocytochemistry, they provided quite a lot
of materials. Website has loads of protocols etc...
Hope this helps

Dr Jonathan Welsh
Cellular and Molecular Medicine
St George's Hospital Medical School
London, UK'

Also, I can highly recommend a book that is most helpful as a starting
point: 'Introduction to Immunocytochemistry' by J.M.Polak and S.Van
Noorden
It contains a number of methods that are easy to follow and also
provides a good selection of practical hints and tips.

As for my question regarding doing double indirect intracellular
labelling, there were a number of respondents who recommended
conjugating primary antibodies to reporter molecules prior to use:

'The Zenon kits of Molecular Probes provide a nice tool to circumvent
some
pitfalls. Please have a look at
http://www.probes.com/products/zenon/zenon.html 
 Best,
Arne'

Hope that helps, thanks everyone,

Sian.




Dr.Sian Rizzo
Prostate Stem Cell Laboratory
Male Urological Cancer Research Centre
Institute of Cancer Research
15 Cotswold Road
Sutton
Surrey SM2 5NG

TEL: +44(0)20 8722 4178
FAX: +44(0)20 8722 4278
email: Sian.Rizzo@icr.ac.uk