Anti cd133 pab

For characterization of primary GBM7 cell culture, the expression of major GBM and cancer stem cell (CSC) markers was assessed. For indirect IF labeling, primary GBM cells were grown in 30-mm Petri dishes to achieve a monolayer. Then, the cells were washed with the phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min, washed, and blocked with 5% goat serum for 30 min. Then, the cell monolayer was incubated with primary antibodies at +37°C for 1 h (dilutions according to the manufacturer’s protocol, Abcam, Cambridge, MA, USA). The following primary antibodies were used: anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (pAb), anti-connexin43 (Cx43) monoclonal antibody (mAb), anti-nestin mAb, anti-CD133 pAb, anti-cyclooxygenase2 (Cox2) mAb, and anti-Ki67 pAb (Abcam, Cambridge, UK). Next, the cell monolayer was washed with PBS and stained by a solution (1:500) of secondary anti-species antibodies: goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 633, 595, or 536 (Abcam, UK) at 37°C for 1 h. After three washes with PBS, cell nuclei were stained with DAPI at a dilution of 1:500 for 5 min, then the cells were embedded in 50% buffered glycerol. Protein expression in primary GBM7 cells was assessed using a confocal microscope (Nikon, Tokyo, Japan).