TEM immunolocalization problems

<< Previous Message | Next Message >>
From:Dwight Beebe <beebed@IRBV.UMontreal.CA> (by way of histonet)
To:histonet <histonet@magicnet.net>
Content-Type:text/plain; charset="us-ascii"

Good morning everyone,
	I have encountered considerable difficulty, i.e., zero success, in
repeating an immunolabelling experiment.  I'm working with LR White
embedded cotyledon tissue, fixed 12/95, probing with a polyclonal Ab
(rabbit), also made in '95.  My secondary is commercial protein A gold.  I
had very specific labelling when the initial experiment was conducted in
'95, but now have no specific label.  I have repeated the previous
successful protocol, but with no luck.  My antibodies have been at -20C,
having only been thawed once to aliquot them into smaller volumes.
	Is it possible that there have been changes in the fixed tissue
that cause loss of epitopes?  I find this hard to believe, but I'd like to
hear from others.  Could the antibodies have lost their specificity?  I
have repeated dilution series experiments, but again, all labellings were
no more specific than buffer controls.  I should say that the antibodies
work fine in Westerns, giving us the same banding pattern we've always seen
with these Ab.
	Any and all comments, criticisms, speculative remarks, etc. would
be most welcome.  Much hinges on the success of the labelling.
	Many thanks,

Dwight Beebe
Prof. Agrege (Associate Prof.)
Institut de recherche en biologie vegetale
Universite de Montreal
4101 est, rue Sherbrooke
Montreal, (Quebec) H1X 2B2 Canada
Tel: 514/872-4563 or -4746 (lab)
FAX: 514/872-9406

<< Previous Message | Next Message >>