Immunofluorescence Staining of GBM Tissues

Cryostat sections of GBM ex vivo tissues were prepared as previously described [3 (link)]. Cells were incubated overnight with the primary antibodies at 4 °C, followed by the secondary antibodies for 1 h at 37 °C. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific; 1:30,000, 30 min, room temperature) and the embedded slides were analyzed by fluorescence microscopy (AxioObserver.Z1; Carl Zeiss AG, Oberkochen, Germany) using the ZEN 3.5 (blue edition) software (Carl Zeiss AG). Used primary antibodies are listed in Supplementary Table S2. If primary antibodies were derived from the same species, non-specific binding was blocked by F(ab) fragments derived from that species (1:1000, from Jackson ImmunoResearch, West Grove, PA, USA). Primary antibodies were omitted for negative controls. Donkey anti-mouse or anti-rabbit IgGs labeled with Alexa Fluor 488 or Alexa Fluor 555 (1:1000; Thermo Fisher Scientific) served as secondary antibodies.