RE: microwave antigen retrieval for PCNA in rats

From:"Su, Phy-Huynh" <psu@shctampa.usf.edu>

Hi Victoria,
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.
Su

> -----Original Message-----
> From:	Victoria Baker [SMTP:vbaker60@yahoo.com]
> Sent:	Wednesday, November 01, 2000 10:15 AM
> To:	HistoNet Server
> Subject:	microwave antigen retrieval for PCNA in rats
> 
> Hi!
> 
> 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
> times
> 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
> minutes.
> 
> 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
> resin.
> 
> 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
> appreciated.
> 
> 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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