problems staining imaginal discs - esp. eye discs

[Anna Levine]

I would be very grateful for help/ideas with the following problem I've been 
experiencing lately:

when trying to stain 3rd-instar imaginal discs using the DAB-HRP method, 
which I have had success with in the past, I now get only a very weak 
signal. This is especially a problem in the eye discs, which I am 
particularly interested in.

I have tried changing almost every component of the method: I've used 
different primary and secondary antibodies, fresh formaldehyde and blocking 
solutions, and new DAB (Sigma Fast DAB tablets; I'd previously used, 
successfully, DAB solution from DAKO). Blocking and antibody dilutions are 
done in buffer containing normal donkey serum - the secondary antibodies are 
raised in donkey, and aliquots of it have given good results in the past.

I've even tried different fly stocks than usual.

It's very difficult to think of what else to try - does anyone have any 
ideas?

Thank you very much in advance!

Anna Levine

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Dr. Anna Levine
Faculty of Life Sciences
Bar-Ilan University
Ramat-Gan 52900
Israel

anna2061 at hotmail.com
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