Anti ng2

Three representative slides from each of the six eyes were used for immunofluorescent staining. Eighteen sections were rinsed with phosphate-buffered saline (PBS) and blocked using DAKO serum-free protein block (X0909; Dako, Carpinteria, CA, USA). The samples were either incubated with primary antibodies at 4℃ overnight, followed by incubation with secondary antibodies for two hours at room temperature the next day, or they were incubated overnight at 4℃ with conjugated antibodies. The following antibodies were used for paraffin sections: Alexa Fluor 488 isolectin GS-IB4 conjugate (1 : 500 dilution; Life Technologies, Carlsbad, CA, USA), Anti-NG2 (1 : 500 dilution; Millipore, Bedford, MA, USA), anti-human CD235 (N-cadherin) (1 : 500 dilution; Biolegend, San Diego, CA, USA), Alexa Fluor 594 goat anti-rabbit IgG (1 : 200 dilution, Life Technologies) and Alexa Fluor 647 donkey anti-mouse IgG (1 : 200 dilution, Life Technologies). Nuclei were counterstained with DAPI (4′,6-diamidino-20 phenylindole). The sections were analyzed with a fluorescence microscope (Eclipse 90i; Nikon, Tokyo, Japan).

Human pericytes, primary astrocytes, and hCMEC/D3 released by 0.025% trypsin/EDTA (Sigma Aldrich, Saint Louis, MO) were resuspended in BBB-working medium (Vasculife-Mv supplemented with 2% normal human serum with antibiotics omitted), and 1.5 ×10³ of each cell type (final volume of 100 μL/well) were seeded onto sterile 1% w/v solid agarose (Sigma) pre-dispensed into low binding 96-well plates (Thermo Fisher Scientific). A further 100 μL of BBB working media (BBBM) was added to bring the volume in each well to a total of 200 μL. Multicellular BBB organoids were allowed to self-assemble (48–72 h) in a humidified 5% CO₂-incubator at 37°C.²² (link)^,30 (link) The orientation of the component cells was determined by pre-staining astrocytes with 1μM CellTrace Far Red (Thermo Fisher Scientific) for 20minat 37°C in the dark prior to assembling organoids. Staining for CD31 on endothelial cells and NG2 on pericytes was performed post-formation. Once organoids formed, they were washed, pooled, and fixed with 3.7% formaldehyde for 10 min prior to staining for 45 min with anti-CD31 (1:200, R&D Systems) and anti-NG2 (1:200, Millipore) post-permeabilization with 0.1% Triton X-100. The receptors were visualized with anti-mouse-IgG Alexa Fluor 450 (CD31) and anti-rabbit-IgG Alexa Flour 488 (NG2). Images were captured with Zeiss LSM 780 and line plot analysis conducted in Fiji.

Tissue sections from all groups were fixed in 4% PFA at room temperature for one hour. They were then washed with PBS, mounted on glass and incubated in a solution of 10% normal goat serum in PBS (MP Biomedical, UK) and 0.25% Triton X-100 (Sigma, Germany) for one hour. Next, the tissues were incubated with 100 µL of a primary antibody (anti-MBP, MBL, anti-NG2 and anti-OX-42; Millipore, Germany) at room temperature for 90 min or in the dark at 4 °C overnight. They were washed three times with PBS and stained with secondary antibodies (Alexa Fluor® 633 and 488, Invitrogen, Germany) that were diluted in PBS (1:100). Of note, a few tissue sections were processed without primary antibodies in order to examine the specificity of immunolabelling with the antibodies, and this resulted in no immunostaining.