A1rmp multiphoton confocal microscope

For tumor microvascular imaging assays, each tumor‐bearing mouse was anesthetized, and 200 μL FITC‐dextran (100 mg/mL, 2000 kDa; Sigma‐Aldrich, St Louis, MO, USA) was i.v. injected for circulating for 2 minutes. Local tumor was scanned and imaged with a Nikon A1R MP⁺ multiphoton confocal microscope (Nikon Instruments Inc., New Orleans, LA, USA). For tumor microvascular perfusion assays, each tumor‐bearing mouse was given an i.v. injection of 200 μL FITC‐lectin (1 mg/mL; Sigma‐Aldrich) 15 minutes before death. Tumors were collected and directly frozen in liquid nitrogen until cryosectioning into 5‐μm sections.

SHG imaging was utilized to evaluate the collagen structure in BHV tissues. Individual BP samples were mounted on an 8-mm Fastwell incubation chamber (Electron Microscopy Sciences) and covered with a 0.17-mm coverslip. SHG microscopy was performed on a Nikon A1RMP multiphoton confocal microscope (Nikon; Minato). The SHG signal was generated using 860-nm excitation light from a Ti:sapphire femtosecond laser and detected using Nikon Apo LWD 25×/NA1.1 gel immersion as an objective with a 400 to 450 nm bandpass filter. All images were acquired at 1,024 × 1,024 resolution using NIS Elements software. For each piece of tissue, SHG imaging was taken on two or three nonoverlapping areas. Collagen alignment analysis was performed using CurveAlign software (https://loci.wisc.edu/software/curvealign), and the alignment coefficient was recorded (N = 12 images per group).

Whole fixed brains of mice that underwent in vivo imaging were clarified using the CUBIC-cancer method as described in Kubota et al. (2017) (link). Briefly, excised brains were delipidated in ½-dH₂0 – diluted CUBIC-L solution (10w%/10w% N-butyldiethanolamine/Triton X-100) for 6 hours followed by incubation in complete CUBIC-L for 5 days with gentle shaking at 37°C. Brains were subsequently washed with PBS and blocked in 10% normal goat serum, 0.5w% Triton X-100 for 6 hours, followed by staining with the indicated primary antibodies (Figures S1C and S1D) for 5 days at room temperature in blocking buffer. After multiple PBS washes, secondary antibody incubations were performed for an additional 4 days at room temperature in blocking buffer. Following final PBS washes, brains were immersed in CUBIC-R solution (45w%/30w% antipyrine/nicotinamide) and stored and imaged in the same solution. High-resolution images of CUBIC-cancer clarified post-mortem brains were acquired using a Nikon A1RMP multiphoton confocal microscope and a 25 × /1.10 NA coverslip-corrected water immersion IR lens objective. For two-photon acquisition, a Chameleon II laser was tuned to 820 nm and images were captured on a spectral detector. Images were taken at 5 μm steps, unmixed and stacks were generated with NIS Elements AR or FIJI/ImageJ software.