[Histonet] Nickel-DAB Signal Fading

[Rene J Buesa]

If when you finished the procedure the signal was strong, it is not a problem with the antibody, it would have reacted with your working concentration, or would not have worked.
I am inclined to think that the nickel solution you use to add to the DAB may have deteriorated in that batch and have caused the fading.
Try some of the block to cut new sections and use a fresh nickel solution to find out what happens in 2 months time.
René J.
 
 

--- On Tue, 2/8/11, Amanda Madden <amkmadden <@t> gmail.com> wrote:


From: Amanda Madden <amkmadden <@t> gmail.com>
Subject: [Histonet] Nickel-DAB Signal Fading
To: histonet <@t> lists.utsouthwestern.edu
Date: Tuesday, February 8, 2011, 6:27 PM


Hello Histonetters!

I am once again completely baffled, and thought you might be able to help. I
run immunocytochemistry on rodent brain tissue every week, always using the
exact same procedures, solutions, buffers, chromagen, and mounting medium.
The only variable is the primary and secondary antibodies. Our protocol has
been time tested and extremely successful. I recently ran a large batch of
tissue, and the staining looked great... except that now, about two months
later, the signal has faded almost to the point of being gone. I am using
Nickel intensified DAB, if that helps, and coverslipping (after an alcohol
series finished with xylene) with Permount. Could it be a problem with the
primary antibody (one which is new to us but has seemed fine in series
dilutions)? Or is it a problem with the chromagen/mounting medium? I have
been storing the slides in slides boxes so I don't think it is light.

Going crazy, and thankful for any advice,
Amanda Madden
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