Af2018

Tumor samples from GBM patients and mouse intracranial GBM models were fixed in 4% paraformaldehyde overnight at 4 °C, followed by overnight cryoprotection with 30% sucrose in PBS at 4 °C. Samples were then sectioned at a thickness of 7 µm. Sections were washed with PBS twice, permeabilized, and then blocked with 0.3% Triton X-100, 5% BSA in PBS for 1 h. Then, the sections were stained with primary antibodies against Iba1 (1 µg/mL, FUJIFILM Wako, #019-19741), CD206/MMR (2 µg/mL, R&D Systems, #AF2535), SOX2 (2 µg/mL, R&D Systems, #AF2018), and CCN1/Cyr61 (10 µg/mL, Novus, #NB100-356) overnight at 4 °C, followed by the secondary antibodies against rabbit or goat immunoglobulin G (IgG) labeled with Alexa Fluor dyes (Thermo Fisher Scientific) at room temperature for 1 h. After immunostaining, the samples were mounted with DAPI Fluoromount-G (SouthernBiotech, #0100-20). Images were obtained under an LSM780 confocal laser scanning microscope (Carl Zeiss).

In preparation for immunocytochemistry, cells were fixed in 4% PFA for 15 min at room temperature and then permeabilised for 1 hr with 0.1% Triton X-100 in PBS. After blocking with 3% BSA, samples were incubated with primary antibodies for 1 h at room temperature or overnight at 4 °C, followed by reaction with fluorescent secondary antibodies for another hour. Antibodies used were rabbit polyclonal anti-active caspase 3 (1:500, Abcam, ab2302), rabbit anti-FGF5 (1:50, Proteintech, 18171-1-AP), goat polyclonal anti-GATA4 (1:200, SantaCruz, sc-1237), chicken polyclonal anti-GFP (1:500, Abcam, ab13970), rabbit polyclonal anti-HSD3B (1:500, gift from Ian. Mason), rat polyclonal anti-Ki67 (1:500, BioLegend, 652401), rabbit polyclonal anti-laminin (1:1000, gift from Harold Erickson), rabbit polyclonal anti-MVH (1:300, Novus Biologicals, NBP2-24558), rabbit polyclonal anti-OSR1 (1:200, Abcam, ab230627), anti-Pax2 (1:200, Novus Biologicals NPB2-57700), goat polyclonal anti-SOX2 (1:200, Novus Biologicals, AF2018), goat anti-SOX9 (1:200, Novus Biologicals, AF3075), rabbit polyclonal anti-SF1/NR5A1 (1:200, Novus Biologicals NPB1-52823), rabbit polyclonal anti-STAR (1:200, Biorbyt, orb7014), rabbit polyclonal anti-STRA8 (1:200, Abcam, ab49602) and rabbit anti-SYCP3 (1:200, Novus Biologicals, NB300-230).