Normal goat serum (ngs)

Isolated vessels were fixed in ice-cold methanol for 10 min at room temperature. After washing with PBS, the vessels were blocked with 10% normal goat serum (Wako) in PBS supplemented with 0.1% Triton-X at room temperature for 1 h. After blocking, the cells were stained with a mouse anti-claudin-5 antibody (diluted 1/50; ThermoFisher Scientific) and a rabbit anti-platelet endothelial cell adhesion molecular-1 (PECAM-1) antibody (diluted 1/50; ThermoFisher Scientific) at 4 ^oC overnight. Then, the cells were washed with PBS and stained with an Alexafluor488-conjugated secondary antibody and an Alexafluor594-conjugated secondary antibody (diluted 1/500; ThermoFisher Scientific). After washing, the nuclei were labeled with 4’, 6-diamidino-2-phenylindol (DAPI; Sigma) for 10 min at room temperature. Images were obtained with a BZ-X700 microscope (KEYENCE).

The cells were fixed with 4% paraformaldehyde for 15 min and treated with PBS containing 2% normal goat serum (Wako), 0.5% BSA (Sigma-Aldrich), and 0.2% Triton X-100 (Wako) for 30 min. Antibodies specific for the following markers were used in this study: SSEA1 (1∶50), SSEA4 (1∶50), TRA1-60 (1∶50), and TRA1-81 (1∶50, ES characterization kit, Merck Millipore, Billerica, MA, USA). The following secondary antibodies were used: Alexa Fluor 488 labeled anti-mouse IgG (1: 200, Merck Millipore), and Alexa Fluor 488 labeled anti-mouse IgM (1∶200, Merck Millipore). For immunostaining for ESC-specific cell markers, we used ESCs characterized previously [14] (link) as positive controls and incubated samples without primary antibodies as negative controls. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (Merck Millipore).

For immunostaining analysis of the cellular localization of OTRs, HEK293T cells co-transfected with an HA-tagged OTR and mSca_mem (10:1 ratio) were fixed in PBS (FUJIFILM Wako) containing 4% (wt/vol) paraformaldehyde for 10 min at room temperature. Next, cells were permeabilized and blocked for 30 min at room temperature in blocking solution (PBS containing 0.2% TritonX-100 and 5% normal goat serum (FUJIFILM Wako)) and then incubated with an anti-HA antibody (rabbit; MBL; 1:2,000 dilution) for 30 min at room temperature. After washing with PBS containing 0.1% TritonX-100 (PBST), samples were incubated with Alexa 488-conjugated anti-rabbit IgG antibody (goat; Jackson ImmunoResearch) for 30 min at room temperature. After three washes with PBST, stained cells in PBS were analyzed by microscopy.

Immunofluorescent staining evaluation was performed with the following standard procedure. After removing the mediums, the cells were washed three times with PBS. The cells were fixed in 4% formaldehyde solution in PBS for 30 min, and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 15 min, followed by blocked with 5% normal goat serum (FUJIFILM Wako Pure Chemical Corp.) in PBS at room temperature for 90 min. The cells were incubated with the primary antibodies at 4℃ overnight, and then incubated with the secondary antibodies at room temperature for 2 h. As the primary and secondary antibodies, mouse anti-Tuj1 (neuronal class III β-tubulin) antibody (R&D Systems, MAB1195) diluted 1:2000 in 1% normal goat serum PBS, and Alexa Fluor 546 goat anti-mouse antibody (Invitrogen, A-11003) at 1:1500 in PBS were used, respectively. DAPI (PromoKine) was used for staining the nucleus. As a negative control, cells on cover glasses without the primary antibody and with the secondary antibody were prepared. The stained cells were observed by a fluorescence microscope, and as for Teas A, representative five separate regions of each substrate were photographed. In the case of Test B, the entire region where the DLC thin films were deposited was photographed, and the experiment was carried out in triplicate.

HASM were cultured in SmGM2 at 37 °C under 5% CO₂. Cells of the passage number between 5 and 6 were used for experiments. Cells were rinsed with Dulbecco’s phosphate-buffered saline (PBS) once and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 min at room temperature, followed by permeabilization with 0.3% TritonX-100 in PBS for 15 min or with 90% chilled methanol for 5 min. After blocking in 5% normal goat serum (FUJIFILM Wako Pure Chemical Corp.) and 0.3% TritonX-100 in PBS, cells were incubated with rabbit polyclonal anti-clathrin heavy chain (1:400) (#ab21679, Abcam) overnight at 4 °C. After washing, cells were incubated with an appropriate Alexa-Fluor-conjugated secondary antibody (Molecular Probes) for 1 h at room temperature.