RNA FISH assays were performed as previously described [17 (link)]. Briefly, complementary probes targeting the “head-to-tail” sequence of circPTEN1 were synthesized by Sangon Biotech (Shanghai, China) (Table S2 ). The cells were cultured on gelatin-coated glass cover slips, fixed with 4% formaldehyde in phosphate-buffered saline (PBS), and permeabilized for 5 min with 0.2% Triton X-100 in PBS. The cover slips were incubated overnight at 37 °C with a hybridization mix containing 1 ng/μL probe mix. The unbound probes were washed off the next day with hybridization buffer, and cells were mounted after staining with DAPI. Images were taken with an immunofluorescence microscope (Nikon A1).
For protein IF assays, cells seeded on cover glass were fixed with 4% PFA for 15 min and washed twice with PBS. Washed cells were permeabilized with 0.2% Triton X-100 for 5 min and blocked for 1 h using 2% BSA dissolved in PBS. The primary antibody was then applied for 3 h at room temperature, followed by three washes in PBS, and fluorophore-conjugated secondary antibody was applied for 1 h. After being washed twice with PBS, cells were stained with DAPI (Sigma). Coverslips were mounted, and cells were evaluated with fluorescence microscopy. A Nikon A1 microscope was used for confocal microscopy.
For protein IF assays, cells seeded on cover glass were fixed with 4% PFA for 15 min and washed twice with PBS. Washed cells were permeabilized with 0.2% Triton X-100 for 5 min and blocked for 1 h using 2% BSA dissolved in PBS. The primary antibody was then applied for 3 h at room temperature, followed by three washes in PBS, and fluorophore-conjugated secondary antibody was applied for 1 h. After being washed twice with PBS, cells were stained with DAPI (Sigma). Coverslips were mounted, and cells were evaluated with fluorescence microscopy. A Nikon A1 microscope was used for confocal microscopy.
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