Bz 800

Cell transfer onto the amnion was performed following previously described methods that are summarized in Fig. 1a 5 (link)6 (link)7 (link)13 (link). Briefly, TEG-coated glass substrate was treated with UV irradiation and the transfer substrate was prepared. Cultured cells (5×10⁵) were seeded onto the transfer substrate (1 cm × 1 cm) and incubated in 5% CO₂ for 3 h at 37 °C. Following the incubation period, the transfer base was placed downward onto the amnion. Growth medium was added and cultured. After 5–18 h incubation, the transfer substrate was gently removed using tweezers. Cells that were transferred to the amniotic membrane were examined under a fluorescence microscope (Keyence BZ800, Keyence, Osaka, Japan) and bright field microscope. In observing transferred cells in vitro, the scaffold (amniotic membrane) was not visualized in figures.

Tissue culture plastic (TCP) was coated with laminin (50 µg mL⁻¹, Sigma-Aldrich L2020), fibronectin (50 µg mL⁻¹, Fisher Scientific CB-40008A), collagen-I (50 µg mL⁻¹, Corning 354236), or collagen-IV (50 µg mL⁻¹, Sigma-Aldrich C0543) solutions for 2 h at 37 °C. Solutions for laminin and fibronectin were dissolved in DPBS; solutions for collagen-I and collagen-IV were dissolved in 0.1% acetic acid. Wells were subsequently washed 3× with DPBS. DIPG spheroids were then placed on coated TCP to assess adhesion and migration. Images were collected with a Keyence BZ-800 with a 10X objective shortly after seeding the DIPG spheroids, 0 h, and at 24 h. To quantify migration, ImageJ was used to measure the migration area at 24 h, which was normalized to the spheroid area at 0 h. For live-cell time-lapse imaging to monitor DIPG adhesion and migration, a DMi8 inverted epifluorescence microscope (Leica) with a 20X objective and equipped with a live-cell incubator was used. Images were captured every 10 or 20 min for 24 h.

The expression level of the intercellular TJ protein zonula occludens-1 (ZO-1) was detected. Nonspecific binding sites were blocked with PBS containing 1% w/v BSA for 30 min at 25°C. The samples were incubated with an anti-ZO-1 antibody (Santa Cruz Biotechnology) overnight at 4°C. The slices were washed several times with PBS, followed by incubation with a tetramethylrhodamine isothiocyanate-conjugated secondary antibody (Santa Cruz Biotechnology) at a ratio of 1:100 for 1 h at 25°C in the dark. Nuclei were stained with DAPI. For the mounting of the samples onto slides, Glycerol was used. Images were acquired with a fluorescence microscope (BZ-800; Keyence, Osaka, Japan).

All rats were handled for 5 days before the experiment. Retrograde tracer was microinjected into spinal dorsal horn^{69 (link)}. After rats were deeply anesthetized with i.p. injection of three types of mixed anesthetic agents (in combination with 0.3 mg kg⁻¹ of medetomidine, 4.0 mg kg⁻¹ of midazolam, and 5.0 mg kg⁻¹ of butorphanol), the spinal cord was exposed by performing a laminectomy over one lumbar segment (between L3 and L5). The dura was cut, subsequently a glass micropipette (tip diameter 20–50 µm) was inserted into the dorsal horn of the spinal cord at an angle of 45 degree to the rostrocaudal axis. Green IX Retrobeads^® (LUMAFLUOR INC., Durham, NC, USA) were bilaterally injected (10 nL, each) using a calibrated injection system (Drummond Nanoject, Broomall, PA, USA). Then, the incision was then closed. Animals were allowed to recover from anesthesia in a warmed box before being returned to the holding cages. Seven days after microinjection, they were perfused and fixed. Brains and spinal cords were collected, and cut into 30 µm of coronal brain and axial spinal sections. Images were scanned by BZ-800 (Keyence, Osaka, Japan) to confirm the existence of Reatrobeads® in the dPVN, LC and DR that were retrograded from the spinal dorsal horn.

For immunocytochemistry, samples (neurospheres or neurons: derived from the 201B7 line) were plated onto poly-L-ornithine/fibronectin-coated chamber slide glasses (Iwaki) and fixed in 4% PFA/PBS for 30 min at room temperature. The slides were rinsed with PBS three times and permeabilized with 0.3% Triton X-100/PBS for 5 min at room temperature. After blocking with Blocking One (Nacalai Tesque, 03953-95) for 15 min at room temperature, the slides were incubated at 4°C overnight with the following antibodies: rabbit anti-α-tubulin (Cell Signaling Technology, 2144; 1:500), mouse anti-β-III tubulin (Sigma-Aldrich, T8660-2ML; 1:500), rabbit anti-tau (Dako, A0024; 1:500), rabbit anti-p38 MAPK (Cell Signaling Technology, 8690; 1:500), rabbit anti-phospho-p38 MAPK (Cell Signaling Technology, 4511; 1:500), rabbit anti-phospho-CDC25B (Thermo Fisher Scientific, PA5-104568; 1:500), and rabbit anti-acetyl-α-tubulin (Lys40) (Cell Signaling Technology, 5335; 1:500). After washing three times with PBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 (Thermo Fisher Scientific, A-11034) or Alexa 555 (Thermo Fisher Scientific, A-21424; 1:500) for 60 min at room temperature and then subjected to nuclear counterstaining with Hoechst 33258 (Sigma-Aldrich, B2883; 10 μg/mL). The samples were analyzed with an all-in-one fluorescence microscope (BZ-700 or BZ-800, Keyence).

Cells were stained with Dextran, Oregon Green 488, 10,000 MW, Anionic (quenched under acidic environment) and Dextran, Tetramethylrhodamine, 10,000 MW, Anionic, Lysine Fixable (pH-independent) (Thermo Fisher) to measure lysosomal acidification. After addition of the two Dextran molecules, cells were incubated for 8 h at 37°C in a glass bottom dish (Iwaki) and incubated overnight after medium change.³⁰ (link) The medium of the labeled cells was replaced with HBSS, and the cells were observed under a fluorescence microscope. The intensity of green and red fluorescence was measured and calculated (BZ-800, Keyence), enabling changes in lysosomal pH to be monitored.