Methods and Techniques Discussion > Immunocytochemistry (ICC)

Non-specific staining of myoblast cell line

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Lot:
We have a problem when staining C2C12 (adherent mouse myoblast cell line) cells grown on slide flasks (4-well from Nunc) with rabbit polyclonals. We use the EnVision system from DAKO and the controls (-primary Ab) show that there is no background staining. The reason we discovered any problems was that we wanted to include "isotype" Ab controls. We chose Abs that were not expected to stain at all, like rabbit anti-GFP and still saw very "nice" staining. Since then we have tried various fixations, buffers and blocking solutions including goat serum, fetal calf serum and BSA at different concentrations. We are going to try blocking with mouse serum and also using a rabbit F(ab)2 nonsense antibody next. In the meantime  - has anybody experienced the same?

Thanks

richard03:
You may need to use higher dilution primary antibody.

ImmunoNYC:
Why not post your protocol and perhaps we can help more then but I agree with Richard that your "very nice" staining may be a result of too much primary. Reduce your concentrations a bit. Also would it be possible that the cells have Fc receptors? Unlikely being fibroblasts but it could explain it. I also found that EnVision is very sensitive to concentration. Anything over 3-5 ug/ml and it gives mega non-specific staining. Must you use EnVision? I usually save that for when I really need a boost.

jayde:
Im currently staining C2C12 for alpha smooth muscle actin which is not supposed to be present in normally cultured cells (According to the literature) however I get really strong stain regardless of treatment (I have literally tried everything).

so although I cant really help, yes I kind of have experience the same thing

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