A1 ti confocal microscope

Cells cultured in confocal dishes were fixed in 4% paraformaldehyde for 20 min and then permeabilized in 0.5% Triton X-100 in PBS for 30 min. After washing with PBS, the cells were blocked with 5% BSA for 60 min. Primary antibodies against USP7 and SOX9 and secondary antibodies [Alexa Fluor 594 goat anti-mouse immunoglobulin G (IgG) and Alexa Fluor 488 goat anti-rabbit IgG, Fudebio] were diluted in 5% BSA. Cells were first incubated with primary antibodies at 4°C overnight and subsequently incubated with secondary antibodies for 1 hour at room temperature. After washing three times in PBS, the cells were incubated in 4′,6-diamidino-2-phenylindole (Beyotime). Representative images were taken using a Nikon A1 Ti confocal microscope (Tokyo, Japan).

For immunofluorescence staining of cryo-sections from mouse samples, aortic sinus was first harvested, washed with PBS and fixed in 4% paraformaldehyde at 4°C for 2 h, followed by dehydration in 30% sucrose solution at 4°C overnight until fully penetrated. Tissues were then embedded in optimum cutting temperature (O.C.T., Sakura, 4583), frozen at -80°C for storage, or cut into 5-μm sections by a Cryostat (Leica CM1950). Cryosections were air-dried for about 30 mins at room temperature, blocked and permeabilized in 5% donkey serum (with 0.1% Triton X-100 in PBS) for 1 h, stained with primary antibodies overnight at 4°C, then incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen, 1:500) for 1 h, followed by DAPI staining and mounting in the anti-fade mounting medium. A Nikon A1 Ti confocal microscope was used to acquire cryo-section, en-face and cell staining images, further analyzed by relevant software. Regions were selected randomly to avoid biasing. For quantification of immunostaining images, at least 3 random fields of view containing the whole artery were counted and the mean values were taken as the data point for each mouse sample. The investigators were blinded to different groups when performing immunostaining and analyzing the data.