microwave antigen retrieval for PCNA in rats

From:Victoria Baker <vbaker60@yahoo.com>


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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