I'm using it to detect oxidative DNA damage in mouse brain (IF experiments) and I obtained a cytoplasmic signal instead of a nuclear signal. Has it been tested to work on mice? I have tried it with two different protocols for saturation, a simple glycine buffer and the MOM kit and the results are quite the same (well, in fact with MOM the signal results are cleaner then with glycine, but it remains cytoplasmic). Has someone had already used it successfully and can give me some advice?
Hi Chiara,
This antibody is species independent as it only detects 8-hydroxy-guanosine, not a specific immunogen sequence, therefore it can be used with any tissue that has DNA or RNA. I don't believe what you are seeing is due to non-specific staining or a mouse-on-mouse issue. The antibody detects 8-hydroxy-guanosine on both DNA and RNA, so it is not unusual to see the detection of RNA in the cytoplasm.
What is your method of membrane permeabilization? If you are not adequately permeabilizing your cells, the antibody may not be able to enter the nucleus to stain the DNA, therefore you may only be left with the detection of 8-hydroxy-guanosine on RNA in the cytoplasm. I would recommend 1% PBST for 30 to 60 minutes. You may still end up with cytoplasmic RNA staining but the staining should be much brighter in the nucleus.
Best of luck!
Have you tried another primary antibody against 8-OHdg? Abcam offers antibodies where the host species is goat. Thereby you would be able to confirm the cytoplasmic staining. As far as I know the 8-OHdg detects DNA damage in general so it might be possible that you are looking at an increase of mitochondrial DNA damage. Especially in oxidative stress related damages this is highly possible. First I would try to confirm the staining with an additional antibody against 8-OHdg and if you can confirm your data you would have to proof the differences by isolating the mitochondrial fraction of your tissue vs the nuclear fraction and do quantitative analysis via ELISA or Westernblot. Good luck!
We tried another ab some times ago, I don't remember who produce it and in what species (anyway it doesn't work at all). I can consider your suggestion about ELISA, but not the western blot: we suppose a general oxidative DNA damage, so we don't have target genes detectable by wb.
Hi Chiara,
This antibody is species independent as it only detects 8-hydroxy-guanosine, not a specific immunogen sequence, therefore it can be used with any tissue that has DNA or RNA. I don't believe what you are seeing is due to non-specific staining or a mouse-on-mouse issue. The antibody detects 8-hydroxy-guanosine on both DNA and RNA, so it is not unusual to see the detection of RNA in the cytoplasm.
What is your method of membrane permeabilization? If you are not adequately permeabilizing your cells, the antibody may not be able to enter the nucleus to stain the DNA, therefore you may only be left with the detection of 8-hydroxy-guanosine on RNA in the cytoplasm. I would recommend 1% PBST for 30 to 60 minutes. You may still end up with cytoplasmic RNA staining but the staining should be much brighter in the nucleus.
Best of luck!
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