Prostate Cell Immunofluorescence Protocol

Cells for immunofluorescence were fixed in ice-cold methanol for 15 min at -20 °C and further treated with 0.1% Triton X-100 for 30 min. Cells were then blocked by 5% BSA for 30 min at room temperature. Primary antibodies were diluted in universal antibody diluent (NCM Biotech) and incubated with prostate cells overnight at 4 °C. The cells were then washed three times with 0.02% TBST, and were incubated with secondary antibodies for 50 min at room temperature followed by wash with 0.02% TBST twice. DAPI was incubated with cells at room temperature for 5 min. Images were acquired with a STEDYCON confocal microscope and processed using Image J and Adobe Photoshop. Quantification of the m⁶A, RBM15, IGF2BP1/2/3 and S9.6 signal intensities was performed according to the previously described method [7 (link)].