Sc 17320

Immunoblotting and immunofluorescent staining were performed as described^{65 (link), 66 (link)}. Specific antibodies against POSTN (Abcam, ab14041, 1: 400), GSC marker SOX2 (Millipore, AB5603, 1:200; Santa Cruz, sc-17320, 1:200) or OLIG2 (R&D systems, AF2418, 1:100), endothelial cell marker Glut1 (Millipore, 07-1401, 1:500) or CD31 (Dako, M082301, 1:100), and macrophage markers Iba1 (Abcam, ab5076, 1:200), CD11b (Abd serotec, MCA711GT, 1:100), Fizz1 (Abcam, ab39626, 1:100), CD163 (Santa Cruz, sc-33560, 1:100), F4/80 (Abd serotec, MCA497RT, 1:100), CX3CR1 (Abcam, ab8021, 1:100) or CCR2 (Abcam, ab32144, 1:100) were used for immunofluorescent staining on GBM tumor sections as indicated. Specific antibodies against POSTN (Abcam, ab14041, 1: 10,000), tubulin (Sigma, T8203, 1:10,000), phosphor-S473 Akt (Cell Signaling Technology, 9271, 1:1,000), Akt (Cell Signaling Technology, 9272, 1:1,000) were used for immunoblot analyses. To determine TAM density, cryosections of GBM tumors were co-stained with Iba1 (or CD11b) and Glut1. The numbers of Iba1⁺ (or CD11b⁺) cells were calculated by ImageJ. Glut1 positive areas were regarded as vessels and determined by ImageJ. For TAMs density in each sample, the number of Iba1⁺ (or CD11⁺) cells was divided by vessel area for normalization to exclude the individual variation in vascularization. The final outcome was defined as the TAM density.