Bx51 imaging system

The lung tissues were fixed in 4% formalin at room temperature for 48 h, followed by paraffin embedding. Serial sections of 5 µm thickness were cut and used for staining. For immunohistochemical staining, each sections were incubated overnight at 4°C with GSDMD primary antibody (1:1,000; cat. no. ab219800; Abcam). The stained sections were observed under a light microscope (Olympus Corporation, Tokyo, Japan) at a magnification of ×100. Five random images per section were collected for analysis.
For immunofluorescent staining, primary antibodies used were ORMDL3 (1:1,000; cat. no. ab211522; Abcam), NLRP3 (1:100; cat. no. ab263899; Abcam). A secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit/mouse IgM antibody (1:1,000; Life Technologies, Inc.), was used. The sections were then incubated with a 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (1:1,000; Dojindo Laboratories, Inc.) for nuclear staining. Digital section images were captured using an Olympus BX51 imaging system (Olympus Corporation) and quantified with Image-Pro Plus (version 6.0; Media Cybernetics).

The aorta root was dissected, embedded and cut into sections at 6 μm. The first section was harvested when the three aortic valve cusps became visible in the lumen of the aorta, and every seventh section was harvested on one slide (10 sections per slide). Lipids were detected using oil-red O staining as previously described.17 (link) Section images were analyzed by an Olympus BX51 imaging system (Olympus, Tokyo, Japan) and quantified with Image-Pro Plus 6.0 software. The atherosclerotic plaque was expressed as a percentage of the total area of the aorta. The protocol of plaque collagen staining was performed as described previously.18 (link) Atherosclerotic plaque collagen was stained by Sirius red and the sections were analyzed by microscopy.