Fluoromount g mounting solution

The D54 and U251 GBM cell lines were cultured on glass coverslips overnight in DMEM/F12 medium supplemented with FBS. Single cells from dissociated xenograft cell cultures were plated on glass coverslips pre-coated with poly-l-ornithine. The coverslips were fixed with a 4% PFA solution for 10 min at room temperature, washed with cold PBS 3 times for 5 min, and permeabilized (PBS with 0.25% Triton X-100) for 10 min. The coverslips were blocked (PBS with 10% donkey serum and 0.1% Tween-20) for 30 min. Primary antibody mouse anti-MMP9 (Abcam, ab119906, Cambridge, United Kingdom) and sheep anti-ChAT (Abcam ab18736) were diluted 1:100 in blocking solution and applied at room temperature for 1 h. The coverslips were washed 5 times for 5 min with PBST (PBS with 0.1% Tween-20). Secondary antibody Donkey anti-Mouse Alexa Fluor 555 (ThermoFisher Scientific) and DAPI diluted in blocking solution were applied at room temperature for 1 h. The coverslips were washed 5 times for 5 min with PBST and then mounted with Fluoromount G Mounting Solution (Electron Microscopy Sciences Cat. #17984-25, Hatfield, PA, USA). Images were acquired using an A1R Nikon laser scanning microscope with a Plan Apo 60×/N.A.1.40 oil objective.