Devices were fixed, permeabilized, and blocked prior to staining. Protein visualization was achieved by staining the fixed devices with anti-CD31 (catalog no. ab3245, Abcam), anti-LRP1 (catalog no. sc-57351, Santa Cruz Biotechnology), and anti–ZO-1 (catalog no. 61-7300, ThermoFisher Scientific) at 1:200 in phosphate-buffered saline (PBS), overnight at 4 °C on a shaker. Secondary antibodies were used at 1:200 in PBS (568 goat anti-rabbit A-11011 or 633 goat anti-mouse A-21052, Invitrogen) and DAPI (D1306, Invitrogen) at 1:1,000. Additional staining details are in SI Appendix, Supplementary Materials and Methods . Images were acquired with a confocal laser-scanning microscope (,pde; FV-1200, Olympus).
For in vivo samples, brains were formalin-fixed and paraffin-embedded before staining with CC-3 (CC3 Rabbit Mab, 1:800; catalog no. 9664L [D175], Cell Signaling Technology) and rabbit polymer secondary (Biocare Medical catalog no. RMR 622L). Quantification of CC3 staining was performed in QuPath version (v)0.2.3 (Queen’s University, Belfast) using QuPath’s build-in “Positive cell detection” (88 (link)) with three ROIs of the same size manually placed per tumor section.
For in vivo samples, brains were formalin-fixed and paraffin-embedded before staining with CC-3 (CC3 Rabbit Mab, 1:800; catalog no. 9664L [D175], Cell Signaling Technology) and rabbit polymer secondary (Biocare Medical catalog no. RMR 622L). Quantification of CC3 staining was performed in QuPath version (v)0.2.3 (Queen’s University, Belfast) using QuPath’s build-in “Positive cell detection” (88 (link)) with three ROIs of the same size manually placed per tumor section.
