RE: microwave antigen retrieval for PCNA in rats
|From:||"Su, Phy-Huynh" <email@example.com>|
I've used PCNA PC10 on swine sections. I've purchased PCNA from Boerhinger
Mannheim, and instead of using microwave antigen retrieval, I've used pepsin
digestion. I couldn't get any staining before enzyme digestion, but got
consistent result after enzyme digestion.
> -----Original Message-----
> From: Victoria Baker [SMTP:firstname.lastname@example.org]
> Sent: Wednesday, November 01, 2000 10:15 AM
> To: HistoNet Server
> Subject: microwave antigen retrieval for PCNA in rats
> We're doing a study with Sprague Dawley rat colons
> that involves doing PCNA staining. I've done PCNA in
> humans and did't have to do any retrieval. Now, I'm
> not all that versed with it in rats. The clone is
> PC10 and It's made by lab vision. I've been told I
> HAVE TO do my retrieval in the microwave and the
> results haven't been consistant at all. We're getting
> high background to no staining. The PI is blaming my
> cap gap stainer, and I know full well that it isn't
> that! They want me to go back to the humidity chamber
> and I'm cringing.
> My protocol is as follows:
> dilution: 1:24k
> control: liver (rat) which stains pretty good most
> retrieval: target from Dako. PL 10 in the microwave
> for 2 minutes. It is just starting to boil at this
> point. 2.5 minutes at PL 2. cooling for 15 to 30
> 3% H2O2 in dH2O for 15 minutes.
> PBS with tween 20 wash for 5 minutes.
> Serum free protein block 7 minutes.
> PCNA 30 minutes at 30 degrees C.
> PBS, 2 washes 5 minutes each (blotting and washing)
> biotinylated 2ndary at 1:500 45 minutes.
> PBS, same as above
> ABC, 15 minutes.
> 3 - 4 buffer washes, 5 minutes each.
> Nova red chromogen 10 minutes.
> wash, counterstain dehydrate clear and mount with
> It would be really appreciated if anyone has any
> input, I don't want to give up my stainer and go back
> to the "stone age" of the humidity chamber with the
> amount of
> immunos I have to do. The stainer by the way is a
> microprobe manual cap gap, so if anyone is using this
> or another type of this you input would also be
> I truly believe my problem is in the retrieval not in
> the stainer.
> Thanks much,
> Vikki Baker
> American Health Foundation
> Valahlla, New York
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