Anti foxd1

After fixing, embedding, sliding, and deparaffinizing, tissue sections were blocked with 3% H₂O₂ and 5% BSA. Then sections were incubated with anti-FOXD1(Invitrogen, Cat No. PA5-35145), anti-KI67 (Proteintech, Cat No. 27309-1-AP), anti-PCNA (Proteintech, Cat No. 10205-2-AP), and anti-GLUT1(abcam, Cat No. ab115730) antibodies at 4 °C overnight. After washing with PBS, immunohistochemical secondary antibody was applied to the sections for 1 h at room temperature, followed by DAB staining, hematoxylin re-staining, and imaging. The results were evaluated blindly by two independent pathologists.

Following antibodies were used for IF and IHC analysis. Anti-USP21 (catalog MA5-34953), anti-FOXD1 (catalog PA5-35145) were purchased from Invitrogen. Anti-ALDH1A3 (catalog ab129815) was purchased from Abcam. Anti-CD44 (catalog 3570) was purchased from Cell Signaling Technology.
GSCs were fixed in 4% formaldehyde for IF staining, permeated with 0.25% Triton X-100, and then blocked with 1% BSA at room temperature for 1 h. The cells were probed with the primary antibody. After washing by PBS-T, cells were combined with the Alexa Fluor 488 (abcam, catalog ab150113) or Alexa Fluor 647 (abcam, catalog ab150079) labeled secondary antibody.We used a mounting medium containing DAPI (abcam, catalog ab104139). The samples were observed through a confocal laser scanning microscope. For IHC staining, the slides were handled as before [29 (link)]. If <10% of the cells in the tumor area were stained, the result would be classified as negative. If the staining were 10% to 100%, the result would be positive. The percentage of positive tumor cells per slide (10–100%) multiplied by the main intensity pattern of staining (1, weak; 2, medium; 3, strong).

Cells were harvested and lysed in RIPA buffer (BOSTER, Cat No. AR0105) supplemented with protease and phosphatase inhibitors for 10 min on ice. Then cellular debris were removed by centrifugation at 12,000 rpm for 15 min at 4 °C and protein concentrations were determined by BCA Kit (BOSTER, Cat No.AR0197). 50 μg of total protein were subjected to denaturing 10% SDS-PAGE, and then transferred to a membrane for subsequent blotting with specific antibodies. Anti-FOXD1(Invitrogen, Cat No. PA5-35145), anti-GLUT1(abcam, Cat No. ab115730), anti-β-actin (Proteintech, Cat No.66009-1-Ig). All the full and uncropped western blots are uploaded as ‘Supplemental Material’.