[Histonet] Re: Antigen retrieval with anti-BrdU

From:"Pixley, Sarah (pixleysk)" (by way of histonet)

Dear Steve:
I believe that your nuclear stain may come from the anti-BrdU and your
detection system rather than your HIER method. Here are the results from
one of my experiments. You can see that the nuclear staining went away
under two conditions: diluting the primary or diluting the secondary and

These were cryostat sectioned slices of mouse noses. The mice had been
perfused with 4% paraformaldehyde, treated with formic acid for
decalcification, then placed in a cryoprotectant containing sucrose, then
frozen and sectioned. After air drying frozen sections, they were postfixed
in 4% para for 10 mins, then 10 mins in hydrogen peroxide in 100% methanol,
then treated with protease K, then blocked with 10% fetal calf serum/0.2%
triton X-100 in PBS. The primary antibody was a sheep anti-BrdU (overnight
at room temp in a humid chamber), the secondary was a biotinylated rabbit
anti-sheep (2 hrs room temp) and the ABC was the Elite version (2 hrs rm
temp). No counterstain was used.

Slide #	Slide Label	1°Ab	2°Ab	ABC	Results
2		1/1000	1/100	1/200	Good label but some Bkg in all
nuclei. More Bkg than slide 5
3		1/5000	1/100	1/200	Beautiful! Dark Cells. No Bkg.
Almost can't see tissue
4		1/1000	1/100	1/500	Good label. Has nuclear Bkg, but
lighter than in slide 2
5		1/1000	1/400	1/200	Good label. More Bkg. Than in slide
4 (prob due to ABC)
6		1/1000	1.400	1/500	Very clean-No nuclear stain

Bold are the concentrations I chose for further exps.
Good luck.
Sarah Pixley,

Has anyone had problems with unwanted specific nuclear staining after
performing heat AR especially from the microwave.  I've been attempting to
stain paraffin embedded rat liver in which a 24hour and 48hour BDL with
Brdu injection 1 hour prior to harvest.  With any form of AR, even limited
80C I get 75-100% nuclear staining.  With no AR I get nothing, not even the
bile ducts.  Does AR tend to effect nuclear staining with some antibodies.
I'm stumped.


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