[Histonet] Re: Antigen retrieval with anti-BrdU
Pixley, Sarah (pixleysk)
PIXLEYSK <@t> UCMAIL.UC.EDU
Mon Oct 17 12:46:02 CDT 2005
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 ABC.
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.
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.
More information about the Histonet