GBM cells were cultured on coverslips in 24-well plates, as previously described [28] (link). Briefly, cultured cells were fixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.1% Triton X-100 for 5 minutes at room temperature. The cells were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. GSCs were fixed as described above; however, the permeabilization step was performed by using 0.3% of Triton X-100 for 20 minutes and then blocked with 5% BSA for 1 hour. The fixed cells were incubated overnight at 4ºC with rabbit anti-SOX-2 (1:400) and with rabbit anti-OCT-4 (1:400) primary antibodies. Then, after the cells were washed with PBS and secondary antibodies staining were performed using goat antirabbit IgG conjugated with Alexa fluor 488 and 546 (1:400) for 2 h, at room temperature. Thereafter, the cells were washed with PBS, stained with DAPI and mounted in Fluoromount-G. Cells were imaged using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany) and analyzed with Leica LAS X Lite.
Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health).
Images were processed using the software ImageJ 1.49v (Wayne Rasband, National Institutes of Health).