Bz 900

Observation of three-dimensional structures was conducted using a Nikon A1R confocal microscope. For long-term observation of the whole culture dish, we used a Keyence BZ-900 with a tiling function. All image analysis was performed using ImageJ [23 (link)] or Fiji [24 (link)]. To observe vessel wall movement in long-term culture, we used the “linear stack alignment with SIFT” plugin for registration of the figures obtained at various time points. We used the “Reslice” command to prepare kymographs of cell movements from time-lapse movies.

Tissues were fixed overnight in 10% formalin, embedded in paraffin, and cut into 5-μm-thick sections for histological examination. The specimens were then deparaffinized for Masson trichrome (MT) staining. For immunohistochemistry, anti-αSMA, anti-CD45, anti-F4/80 and anti-CD3 antibodies were obtained from BD Biosciences (NJ). Nuclei were counterstained using DAPI (Life Technologies, CA). Fluorescence was observed using a fluorescence microscope equipped with the appropriate filter sets (BZ-900, KEYENCE, Osaka, Japan). Areas of interest were quantified using a BZ-H3C module (KEYENCE).

For immunocytochemistry, HDBECs were incubated with A594-conjugated human IgG (Cat. 009-580-003, 10 μg ml⁻¹; Jackson ImmunoResearch, West Grove, PA) for 1 h, fixed with 4% PFA for 1 h at 4 °C, permeabilized with 0.1% triton-X100 in 1% bovine serum albumin/PBS, and blocked with Image-iT FX signal enhancer (Invitrogen) before the staining. Subsequently, cells were incubated with anti-EEA-1 Ab (Cat.2411, Cell Signaling Technology, Danvers, MA) at room temperature for 1 h, washed and incubated with anti-rabbit IgG A488 dye (Invitrogen) for 30 min at room temperature, and mounted with ProLong Diamond Antifade Mountant with DAPI (Life Technologies). Alternatively, cells were incubated with HCS CellMask™ Green Stain (Cat. H32714, Invitrogen) at 2 μg ml⁻¹ for 30 min at room temperature, and mounted. Otherwise, cells were stained with anti-caveolin 1 Ab (Cat. ab2910, Abcam) or anti-clathrin Ab (Cat. ab21679, Abcam) overnight at 4 °C, incubated with anti-rabbit IgG A488 dye (Invitrogen) for 30 min at room temperature on the following day, and mounted. For the acid wash^{34 (link)}, cells were washed with 0.2 M acetic acid in 150 mM NaCl at 4 °C after the IgG endocytosis assay and before the fixation. These slides were observed under a fluorescent microscope, BZ-900 (KEYENCE).

A total of 5 × 10⁴ cells were seeded on sterile coverslips into 24-well plates and cultured for 72 h. Cells were washed with PBS containing MgCl₂ and CaCl₂ (PBS (+/+)) and fixed for 20 min at RT with a 3.7% formaldehyde solution. After three washing steps and blocking for 1 h at RT with 1% BSA (v/v)/ PBS (+/+), cells were stained with an anti-ZO-1 antibody for 4 h at RT (monoclonal rabbit IgG, diluted 1:150 in incubation buffer (0.3% Tween-20, 0.1% BSA in HEPES); #8193, Cell Signaling Technology, Danvers, MA, USA). Washing steps (3 × 5 min with PBS (+/+)) were followed by incubation with a secondary antibody (polyclonal goat anti-rabbit FITC, 1:250 in incubation buffer (0.3% Tween-20, 0.1% BSA in HEPES)) for 1 h at RT in the dark. Nuclei were stained with DAPI (#9542, Sigma Aldrich, St. Louis, MO, USA, 1:1000 in PBS (+/+), 10 min, RT, dark). Coverslips were transferred to glass slides and mounted with Fluoromount-G^TM (#00-4958-02, Invitrogen, Waltham, MA, USA). Slides were stored at −20 °C in the dark and imaged with a fluorescence microscope (Keyence BZ-900, Osaka, Japan).