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

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From:Amos Brooks <> (by way of Marvin Hanna)
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Hi Sandy,
    You didn't mention how long you have your CD3s in primary. With the
LSAB2 system it should be
under 20 minutes. If it is 20 minutes, try 10. And despite what any vendor
says about how clean
their detection kits are, protein block is sometimes necessary.
    Also how are you fixing your specimen? If there is Prefer fixative
involved expect problems
even if it is a secondary fixative (received in formalin, processed or held
in Prefer). Often the
fixative can cause background staining.
    Regardless, I would consider reducing the time in primary to tweak it
out and possibly
diluting the predilute further. We routinely do this with our CAM 5.2 which
is purchased as a
predilute according to the manufacturer (whose name eludes me at the
moment) in spite of a rather
short incubation time. Try 1:2 to 1:5 if it comes down to this.
    Fortunately this may be a temporary thing with you. Occasionally there
is lot to lot
variability in staining intensity. That is why it is a good idea to test
each new lot when it
arrives. I think it is required by CAP too. (College of American
Pathologists) If nothing else it
keeps your good antibodies good and may improve your not so good ones too
(we all have them :-) ).

good luck
Amos Brooks

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

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