Ab76245

After treatment, BV-2 cells were fixed with 4% paraformaldehyde for 20 min then permeated by 0.3% Triton X-100 for 10 min and subsequently blocked by 1% BSA for 20 min. Primary antibodies (galectin-3, ab76245, Abcam, 1:400; lysosomal associated membrane protein 1, LAMP1, ab208943, Abcam, 1:400; Lipid A, GTX40001, GeneTex, 1:400; cytosolic phospholipase A2-α, cPLA2, sc-454, Santa Cruz, 1:100; Cyt c, 66264–1, Thermo Fisher, 1:400) were incubated with cells overnight at 4°C. Fluorescence conjugated secondary antibodies (Jackson ImmunoResearch, 711-025-152, 705-095-147, 115-095-003, 715-585-150, 1:200) were added at room temperature for one hour.
After fixing with 4% paraformaldehyde, cells were stained with BODIPY™ 493/503 (Invitrogen, Life Technologies, Carlsbad, CA, USA) or PI (Beyotime, Shanghai, China) for 20 min at room temperature. TUNEL staining (MK1027, BOSTER, Wuhan, China) was performed following the manufacturer’s instructions.
Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI), and all images were captured with fluorescent microscopy (Olympus BX41).

For cultured BMDM staining, the primary antibodies were as follows: Ki67 (NB500‐170, Novus Biologicals, Littleton, CO) and Galectin‐3 (Mac2) (ab76245, Abcam, Cambridge, MA). For staining of aortic root sections, the following primary antibodies were used: EGF‐like module‐containing mucin‐like hormone receptor‐like 1 (F4/80) (PA5‐32399, Invitrogen), iNOS (ab3523, Abcam), CD206 (ab64693, Abcam), cleaved caspase‐3 (9661, Cell Signaling Technology (CST), Danvers, MA), CD68 (ab125212, Abcam), α‐actinin (ab137346, Abcam), CD3 (ab16669, Abcam), and Irf1 (8478, CST). The goat antirabbit secondary antibodies Alexa Fluor 647 (red; ab150115, Abcam) and Alexa Fluor 488 (green; ab150077, Abcam) were employed for immunofluorescent staining. A TUNEL Assay Kit (G3250, Promega, Madison, WI) was used to evaluate apoptosis levels in cultured BMDMs and aortic root sections. Cultured BMDMs and aortic root sections were also subjected to 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma) staining to identify nuclei. Images were captured using a RVL‐100 microscope.

The harvested xenograft tumor tissues were paraffined and sectioned, and the sections were de-paraffined and rehydrated. After antigen retrieval using a citrate buffer solution in a microwave and endogenous peroxidase blockage using a 3% hydrogen peroxide, the slides were further blocked with 5% goat serum at room temperature for 2 h. Subsequently, the sections were incubated with the antibodies of Ki67 (1:1000, ab15580, Abcam), GAL3 (1:2000, ab76245, Abcam), CD163 (1:200, ab182422, Abcam), and CD206 (1:500, #24595, CST) overnight at 4 °C, followed by incubation with rabbit anti-mouse IgG (1:1000, #58802S, CST) or goat anti-rabbit IgG (1:2000, 7074, CST) at room temperature for 1 h. After color development using DAB, the sections were counter-stained with hematoxylin, dehydrated with ethanol, cleared with xylene, and mounted with a neutral mountant under a light microscope. The percentage of positive cells under the view, stained in brownish, was then calculated.