Goat serum

Brains were prepared 48 h post adoptive cell transfer. Brains were embedded in Tissue-Tek (Sakura) immediately after explantation and cut into 100 µm thick coronal tissue slices and stored at -80°C. Three sections per brain in 500 µm distance were stained and analyzed as follows. Sections were dried at RT for 30 min, fixed in ethanol-acetone (1:1, Sigma) for 10 min at RT, washed 3 times 5 min in PBS (PAN Biotech), blocked in PBS (PAN Biotech) + BSA (1%; PAN Biotech) + goat serum (10%; Sigma)+ tween20 (0.2%; Sigma) for 50 min at RT. Then sections were stained for 120 min at RT in PBS + BSA + goat serum + tween20 + primary antibody (rat anti CD31, BD; 1:100), washed 3 times 5 min in PBS and stained in PBS + 1% BSA + 10% goat serum + 0.2% tween20 + secondary antibody (donkey anti rat-Alexa647, life technologies; 1:100) for 60 min at RT in the dark. After washing 3 times in PBS (PAN Biotech), DAPI staining (1 µg/ml; Sigma) was performed for 10 at RT and the sections were embedded in fluorescent mounting media (Dako).

Passage 3 (P3) KSCs were fixed in 4% paraformaldehyde/PBS buffer in a 24-well plate (Corning Inc., USA) at room temperature, permeabilized with 0.1% Triton X-100 (Beijing Chemical Factory), blocked for 30 min in 10% goat serum (BD, USA), and then incubated with primary antibodies at 4°C overnight. The primary antibodies included mouse anti-E-cadherin polyclonal antibody, rabbit anti-ZO-1 polyclonal antibody, rabbit anti-fibronectin polyclonal antibody (Santa Cruz, USA), mouse anti-N-cadherin polyclonal antibody, mouse anti-α-SMA polyclonal antibody (Sigma, USA), and rabbit anti-Vimentin polyclonal antibody (Cell Signaling Technology, USA). The secondary antibodies—tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit IgG, goat anti-mouse IgG, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG, or goat anti-mouse IgG (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., China)—and 4′-6-diamidino-2-phenylindole (DAPI, BD, USA) were added sequentially. Imaging was performed with an IX70 confocal fluorescence microscope (Olympus, Japan).