C2 plus microscope

Endothelial cell proteins were stained with the relevant antibodies, followed by incubation with cy3-labeled goat anti-rabbit fluorescent secondary antibody, staining with DAPI, and images were obtained in a Nikon C2 Plus microscope (Nikon, Tokyo, Japan) using C2 plus software, with the same conditions of exposure and excitation for all images (DAPI: Emission wavelength 447.0, Excitation wavelength 408.0, Pinhole radius 60, Alx546: Emission wavelength 585.0, Excitation wavelength 543.5, Pinhole radius 60), and randomly selected images in each group were compared.
Antibody dilution: ZO-1 (1:50; Thermo Scientific, Beijing, China), Occludin (1:50; Thermo Scientific, Beijing, China), Claudin-5 (1:20; Thermo Scientific, Beijing, China). Details are provided in our prior studies (He et al., 2020 (link); Ning et al., 2022 ). ZO-1, Occludin, and Claudin-5 are red. Nuclei are blue. Scale bar = 30 μm.

HCC827/hGFP1-10 cells (25,000 cells/well) were grown overnight in LabTek II CC2 glass chamber slides (Nalge Nunc International) then transfected with DNA (400 ng/well). After 24 h cells were fixed, permeabilized and blocked using the Image-iT Fixation/Permeabilization Kit (Molecular Probes). After blocking, cells were stained for β-Actin and nuclei using ActinRed and NucBlue staining kits (Molecular Probes), washed 3 times with PBS and mounted with ProlongDiamond Antifade (Molecular Probes). Fluorescence images were acquired on a Nikon C2 plus microscope (standard FITC, TRITC and UV fluorescence filters) through a 20x DIC objective and using NIS Elements software. Exposure times for each channel were kept constant between each sample: Nuclei (UV) 600 ms, GFP (FITC) 300 ms, ActinRed (TRITC) 200 ms.