I performed immunodetection of modified nucleotides (5mC and 8dG) on neuronal and astroglial primary culture, and I expected to observe the fluorescence signal only from nuclei, whereas the signal is much more intensive in cytoplasm than in the nuclei.
My question is if this situation is normal and I observe the epitranscriptomical modifications or the protocol for protein immunodetection is not suitable for modified nucleotides immunodetection? Cells were fixed with 4% PFA in PBS and then permeabilized with 0,3% Triton X-100 in PBS for 15 minutes. Primary antibodies were added for 8 h in 4*C, then cultures were washed and the secondary antibodies were added for 1 h in RT.
Przemysław Duda ....what does your negative control look like? How long was your fixation? Did you perform any other controls that would help figure out this issue?
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