Ab210826

The harvested aortic roots were embedded and sliced. Quantitative immunostaining was performed for macrophages, vascular endothelial cells, and smooth muscle cells in frozen slides of the aorta using an anti-CD68 antibody (ab53444, Abcam, USA), anti-α-SMA antibody (ab7817, Abcam, USA), anti-TXNIP antibody (ab210826, Abcam, USA), and anti-CD31 antibody (ab124432, Abcam, USA). Macrophage, smooth muscle cell, vascular endothelial cell, and TXNIP staining was expressed as the fluorescence intensity of the CD68-, α-SMA-, CD31-, and TXNIP-positive areas respectively.

The primary liver macrophages were rinsed with PBS and detached with trypsin. The detachment was terminated with medium containing serum. Two tubes (3 × 10⁵ cells/tube) of cell suspension were prepared. One tube was added with 1 μl of phycoerythrin Cy5-conjugated anti-mouse monoclonal antibody to F4/80 (ab6640, Abcam Inc., Cambridge, MA, U.S.A.) and another tube was added with 1 μl of phycoerythrin Cy5-conjugated anti-mouse monoclonal antibody to IgG2b (ab210826, Abcam Inc., Cambridge, MA, U.S.A.) as control. After incubation for 30 min at 4°C with the avoidance of light, the excess unbound antibody was washed off with PBS containing 5% FBS. The cells were re-suspended in 500 μl of sheath solution and measured by a flow cytometer (FC500, Beckman Coulter, Inc., Chaska, MN, U.S.A.) within 1 h.

Fluorescence staining was performed as described previously (Zhang et al., 2019a (link)). Briefly, SFs were seeded on glass slides, fixed with 4% PFA for 30  min, and then permeabilized with 0.2% Triton X-100 for 15 min. Then, goat serum was used to block non-specific antigens on each slide. D-Hanks was used to rinse the samples, which were incubated with primary antibodies against TXNIP (1:250, ab210826, Abcam), anti-NLRP3 (ET1610-93, 1:250, HUABIO) or IL-1β (1:100, A10609, ABclonal) in cold storage overnight. The glass plates were rinsed the next day and then incubated with CoraLite594-or Fluor488-conjugated secondary antibodies (1:250) at room temperature in the dark, followed by labeling with Antifade Mounting Medium with DAPI for 10 min. The images were observed and captured with an LSM700 laser confocal microscope (Zeiss, Germany). The average fluorescence intensity of the cells was determined using Image-Pro Plus software (Media Cybernetics Inc.).