Ab107595

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.

Immunophenotyping of the cells for surface and intracellular markers was performed upon reaching 80% confluence of the cultures. The harvested cells were centrifuged at 800g for 10 min, the supernatant was discarded, and then the cells were fixed in 2% paraformaldehyde for 15 min at room temperature (RT), diluted with 5 ml of PBS, and centrifuged at 1,500g for 10 min. The pellet was then resuspended in 1 ml of PBS. For immunostaining, 1 × 10⁵ fixed cells were incubated in 100 μl of Rinsing Solution (Miltenyi Biotec, Somerville, MA, USA) with primary antibodies to CD90 (ab225, 1/100, Abcam), CD45 (130-107-846, clone REA504, 1/20, Miltenyi Biotec, Somerville, MA, USA), CD105 (ab107595, 1/100, Abcam), and CD34 (PAB18289, 1/100, Abnova) at RT for 1 h. Subsequently, the cells were incubated with secondary antibodies: anti-mouse Ig-FITC (ab6785, 1/500, Abcam) or anti-rabbit Ig-PE (sc-3739, 1/100, Santa Cruz) at RT for 1 h in the dark. After the incubation, the cells were washed in PBS, resuspended in 0.5 ml PBS, and then transferred to fresh tubes for analysis using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with CellQuest software.

Immunohistochemistry and immunofluorescence analyses were performed according to the standard protocol. We incubated the sections with primary antibodies against: type 2 collagen (Col 2) (ab34712, abcam, UK), CD105 (ab107595, abcam, UK, or ab11414, abcam, UK), proliferating cell nuclear antigen (PCNA) (ab29, abcam, UK), Ki67 (ab15580, abcam, UK), SOX9 (ab185966, abcam, UK), or P53 (ab131442, abcam, UK) overnight at 4 °C. The details of the primary antibodies were listed in the supplementary materials (see Additional file 5). For immunohistochemistry analysis, we used the MaxvisionTM2 HRP-Polymer anti-Mouse/Rabbit IHC Kit (KIT-5920, Maixin, China) to perform DAB staining and the sections were counterstained with hematoxylin. For immunofluorescence analysis, the sections were incubated with Alexa Fluor 594-preadsorbed goat anti-rabbit IgG (1:300, ab150084, abcam, UK) and Alexa Fluor 488-preadsorbed goat anti-mouse IgG (1:200, ab150117, abcam, UK) for 1 h at room temperature. The DAPI Fluoromount-G (0100-20, SouthernBiotech, USA) was used to mount the sections. The staining intensity of the immunohistochemical images were quantified by Image J (NIH, Bethesda, USA). As for immunofluorescence analysis, the double staining-positive cells (yellow) were counted. Three fields were randomly selected per section. Quantitative analysis was performed by three independent evaluators.