re: immuno staining queries

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From:"Nicola Falconer" <>
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Dear all

Anyone not interested in immuno scroll down now. There have been some immuno
related issues raised recently which I would like to add my 2 cents
 English key board doesn't have a cents sign) worth to.

Keith Ryan in Plymouth, asked about general tips for immuno. For each new
antibody, irrespective of what manufactures say, try as many different forms
of antigen retrieval as you can. Titrate the antibody out to give strong
positive staining WITHOUT background, this is usually more dilute than the
manufacturer's recommendation, even with prediluted antibodies, using too
strong a dilution is the most common cause of background staining. Wash well
between stages.

Tibor & Juan in Scandinavia, again wash well, there is nothing wrong with
using a PAN (PAP ?) pen as it will help to localise the reagents but you
must wash well.

Lynn Gardner asked about immuno on decaled tissues. There are no extra
problems provided you do not decal in something horrid like conc. nitric
acid. EDTA or 5 - 10% formic acid will allow immunostaing as normal with
most antibodies.

Bob Richmond & Patsy Ruegg both commented on problems with heat mediated
antigen retrieval, again this is not a universal antigen retrieval
technique. Pick the one that works best for EACH antibody on your tissues.
Just because one method works well for one antibody does not mean it will
work for another even if they are similar, different clones of the same
antibody can require different retrieval methods.

Immuno does not always follow rules of logic and consistency, and with
apologies to Hill St Blues " Remember it's tough on the immuno bench".

sorry for the long post

John Auld
Royal Free Hospital

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