Some time ago, I wanted to perform immunofluorescence on the S protein of mouse CD4 T cells using a Leica super-resolution microscope. I fixed with 4% paraformaldehyde, then added it to the well plate that had been coated with 0.1mg/ml poly-L-lysine, and then proceeded with the routine IF operation. However, under the microscope, there were many fluorescent particles in the background, and different focal planes showed different results (for example, the S protein was inside the nucleus, and after adjusting the focal plane, it appeared around the nucleus).
Dear Ke Zhao,
I think you need to check your cells for mycoplasma contamination. The simplest method is DAPI staining. If you find small luminous dots in the cytoplasm of the cells, then this is mycoplasma.
Best regards,
Rustem Uzbekov
Thank you for your response, but I don't think it could be mycoplasma contamination. Since I have already performed DAPI staining, and the fluorescent particles are from the staining of another protein. For the suspension cells' immunofluorescence, I think the main concern is how to naturally adhere to the wall while reducing background noise.@Rustem E Uzbekov
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