Hi everyone, I am working on BrdU labeling and immunostaining recently. I have tried HCl hydrolysis, it didn’t work so I used 60 degree overnight incubation in citric acid ph6 to denature DNA, slides were allowed to slowly cool down to room temperature after that. With this method, BrdU staining are clear and bright. but sometimes, when BrdU were administered at low dose ( or in pulse chase experiment, Brdu were diluted because of proliferation) I can hardly distinguish them from the background.
How can I improve the signal intensity?
I suppose the DNA desaturation step is the key, to expose the epitope in chromosome completely, should I let the slides cool down instantly (to avoid the double strand come together again?)?
I am also trying to increase the temperature of HIER, but my tissue section will fall off from slides if incubated at over 80 degree.
I suggest 1%HCl+1%EDTA mexture for hydrolysis step.
It will be useful for DNA denatured
I suggest 1%HCl+1%EDTA mexture for hydrolysis step.
It will be useful for DNA denatured
I would strongly recommend to switch to EdU staining and azide-labeled fluorophores which does not require DNA denaturation. There are commercial kits available or you can purchase the reagents separately.
If you have to stick to BrdU, here is denaturation protocol I used before, which actually worked quite well.
Add denaturing solution (2M HCl) and incubate for 20min at RT
Discard supernatant
Wash once with wash buffer (PBS + 0,5%BSA)
Add 0,1M sodium tetraborate, pH8,5 and incubate for 2 min at RT
Wash again with wash buffer and proceed with antobody staining
Good luck!
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