Cryomount 1

The TJ barrier function was assessed in vivo using a previously described biotinylation technique²⁶, ²⁷, ²⁸ with some modifications. First, 50 μL of 10 mg/mL EZ‐Link Sulfo‐NHS‐LC‐Biotin (21355; Thermo Fisher Scientific) in PBS containing 1‐mM CaCl₂ (C‐34006; PromoCell, Heidelberg, Germany) was injected by Myjector syringe (SS‐05M2913; Terumo Corporation, Tokyo, Japan) into the dorsal ear of the C57BL/6J mouse under anaesthesia. After 60 min, we sacrificed the mouse and collected ear samples. In the 3D model, the insert was turned upside down and the tracer solution was applied to the bottom side. Sixty‐minutes later, cell sheets were collected. We soaked in vitro and in vivo samples in a frozen section embedding agent (Cryomount I, 33351; Muto Pure Chemicals) and sectioned them with a cryostat (Leica CM1950, Leica Biosystems). The staining procedure was performed as described above, except that streptavidin Texas Red (dilution 1:200 in PBS 189738; Merck, Darmstadt, Germany) was additionally mixed into the solution of secondary antibodies. Fluorescence microscopic images were taken (BZ9000, BioRevo; Keyence).

Skin and buccal mucosa specimens were frozen in an optimal cutting temperature (OCT) compound (Cryomount I, 33351; Muto Pure Chemicals, Japan) and sectioned with a cryostat (Leica CM1950, Leica Biosystems, Germany). To prepare horizontal‐sliced sections, specimens were embedded horizontally in the OCT compound, and 5 μm‐thick sections were prepared. Frozen sections were incubated with primary antibodies: rabbit anti‐ZO‐1 (dilution 1:200 in PBS, ab2168; Abcam, UK), rabbit anti‐claudin‐1 (dilution 1:200 in PBS, ab15098; Abcam, UK), rabbit anti‐occludin (dilution 1:100 in PBS, 71‐1500; Invitrogen, CA), mouse anti‐keratin14 (dilution 1:200 in PBS, ab7800; Abcam, UK), rabbit anti‐keratin10 (dilution 1:500 in PBS, 905404; BioLegend, CA), rabbit anti‐cleaved caspase‐3 (dilution 1:100 in PBS, #9661 S, Cell Signalling Technology, MA) or anti‐Ki‐67 (dilution 1: 200, NB600‐152, Novus Biologicals) at 37°C for 60 min. The sections were subsequently incubated with Fluorescein isothiocyanate (FITC)‐conjugated goat anti‐rabbit IgG H + L (dilution 1:200 in PBS, 234; MBL), alexa 488‐conjugated anti‐mouse IgG3 (dilution 1:1000 in PBS) or alexa 568‐conjugated anti‐rabbit IgG (dillution 1:1000 in PBS) at 37°C for 60 min and mounted in a Pharma Fluor aqueous mounting medium (TA‐006‐FM; Thermo Fisher Scientific, MA). The stained immunofluorescent samples were observed with a BZ9000 microscope.

Mice were perfused with PBS and then with 4% paraformaldehyde (PFA) in PBS and the spinal cords (L2–L5) were fixed with 4% PFA in PBS for 1 day. The lumbar spinal cords were then incubated in 30% sucrose for 24 h, embedded in CryoMount I (Muto PureChemicals), and frozen sections (40 μm) were produced. Nissl stained were performed using 0.1% cresyl violet acetate (WALDECK). Motoneurons showing a clear nucleolus and distinctly labeled cytoplasm with neuronal morphology, with cell bodies >20 μm in diameter and located in the anterior horn, were included in cell counts. All motoneuron counts were performed in a blinded fashion. For the immunohistochemistry, frozen sections were blocked with 1% BSA at room temperature, and each section was incubated with the primary antibodies. After three washes with PBS, sections were incubated with the secondary antibody solution at room temperature. All images were obtained using a Leica TCS SP5 confocal laser scanning microscope.