RE: CD3 has too much background, ca you help?

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here, here Bob, i don't do any hier anymore, if i do anything at all i use
enzyme digestion, it is much easier to control.
patsy ruegg

-----Original Message-----
From: Robert Schoonhoven []
Sent: Friday, January 28, 2000 8:19 AM
To: Sandy Julsing
Subject: Re: CD3 has too much background, ca you help?

Not all tissues or antibodies need antigen retrieval.  Try it without
the retrieval procedure or try using one of the enzyme treatments eg:
pepsin.  I would do both.

Over the past few years there have been some great things to happen in
IHC and the various heat induced methods of antigen retrieval are some
of them, BUT ... IT IS NOT ALWAYS NECESSARY (sorry to shout but I have
run into this so many times while consulting.....) to use them and they
can cause non-specific background staining and false positives.  Usage
of these techniques is dependent not just on the antibody but on tissue,
fixation and processing as well (not to mention the phase of the moon
and what side of the bed you got up on).

Sandy Julsing wrote:
> Histo-buddies
>      Our CD3 has too much background stain including in the gastric
glands. Our protocol is...
> -DAKO pre-dilute CD3
> -antigen retrieval with citrate buffer pH 5.8-6.2 for 20 min with a 20 min
cool down
> -LSAB2 detection kit 10min/10min/5min
> -Stained on the DAKO autostainer
>  Any help would be greatly appreciated, thanks,  Sandosis

best regards,
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis and Mutagenesis
Dept. of Environmental Sciences and Engineering
University of North Carolina
Chapel Hill, NC 27599
office 919-966-6343
   Lab 919-966-6140
   Fax 919-966-6123

Don't go around saying the world owes you a living; the world owes you
nothing; it was here first.
Mark Twain [Samuel Langhornne Clemens] (1835-1910)

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