Ab154193

The subcutaneous xenografts from all mouse groups were extracted in the sixth week following transplantation, and the gross morphology and texture of the xenografts were visually assessed. The xenografts were preserved with 4% paraformaldehyde at 4°C overnight. HE and immunohistochemical staining (CD31, VEGFA, CD105) were performed according to the kit guidelines. HE staining was used to determine the existence of a hydrogel within the xenograft, the absence of the hydrogel grid structure, and the presence of tumor cells within the hydrogel. Immunohistochemical staining for CD31 (anti-human/mouse CD31 (ab28364), Abcam) and VEGFA (anti-human/mouse VEGFA (ab52917), Abcam) was employed to assess neovascularization in the xenograft. CD105 (anti-human CD105 (ab114052) and anti-human/mouse CD105 (ab107595), both from Abcam) were used to evaluate the vascular component of the xenografts. In this research, the source of neovascularization in the xenografts was examined by double immunofluorescence staining for vWF (rabbit anti-human vWF (ab154193), Abcam) and glial fibrillary acidic protein (GFAP) (mouse anti-human GFAP (MAB2594), R&D Biosystems), following the manufacturer’s guidance.

All subcutaneous xenograft tumors were obtained at 6 weeks after transplantation and fixed with 4% paraformaldehyde at 4°C overnight. Samples were stained with hematoxylin and eosin according to the instructions to analyze the presence of hydrogel and glioma cells within xenograft tumors. Immunohistochemical staining was performed with primary antibodies (anti-human/mouse CD31 (ab28364), anti-human CD105 (ab114052), anti-human/mouse CD105 (ab107595), all form abcam) following the manufacturer’s instructions. Immunofluorescence staining was carried out using primary antibodies (rabbit anti-human vWF (ab154193) form abcam, mouse anti-human GFAP (MAB2594) form R&D Biosystems) according to the protocol. In this study, CD31 was used to detect the neovascularization within xenograft tumors and CD105 was used to evaluate the composition of neovascularization. Particularly, vWF/GFAP double immunofluorescence staining was used to evaluate the origin of neovascularization.

Immunofluorescence staining was used to determine the location of CXCR7. Briefly, the tissue sections were fixed with 4% paraformaldehyde for 30 min. After treatment with 0.6% Triton X‐100 for 1 hr, the sections were blocked with donkey serum. Then, the sections were incubated overnight at 4°C with primary antibodies including anti‐CXCR7 (1:200, Abcam, ab137485) and anti‐vWF (1:200, Abcam, ab154193), followed by incubating with Alexa Fluor 488‐ or 568‐conjugated secondary antibody was added (1:500, Life Technologies). Samples were then co‐stained with DAPI (Sigma‐Aldrich) for 15 min at room temperature. Cover glasses were mounted with aqueous mounting medium (Sigma‐Aldrich, F4680). The images were obtained using a Zeiss Live Cell Imaging System.