Anti cd86

Immunofluorescence staining was performed as previously described with minor modification [28 (link)] [PMID: 33,472,663]. The lung tissue or THP-1 cell sections were blocked with 1% bovine serum albumin (BSA) (Sangon Biotech, Shanghai, China) for 15 min and incubated with anti-CD68 (diluted 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-CD86 (diluted 1:100; ABclonal Technology, Wuhan, China)), anti-iNOS (diluted 1:100; Affinity Biosciences, Changzhou, China), anti-NLRP3 (diluted 1:100; ABclonal Technology, Wuhan, China), anti-ASC (diluted 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-p65 (diluted 1:400; CST, Danvers, MA, USA) overnight. Subsequently, the slices were incubated with corresponding secondary antibodies (Cy3: goat-anti rabbit, diluted 1:200, Invitrogen, Carlsbad, CA, USA; FITC: goat-anti-mouse, diluted 1:200, Abcam, Cambridge, UK), followed by counterstaining with 4’, 6-diamidino-2-phenylindole (DAPI) (Aladdin, Shanghai, China). Fluorescence microscope (Olympus, Tokyo, Japan) was used to capture stained images.

We seeded RAW264.7 cells on 24-well plates (1 × 10⁵ cells/well) and used BSHXD and LPS to process for a certain time. Then we fixed the cells in the pore plate and incubated overnight at 4 °C with anti-CD86 (1:1000, A1199, Abclonal, Wuhan, China) antibody. The Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) (1:1000, ab150114, Abcam) were incubated with cell for 1 h at 37 °C.100 μl Alexa Fluor® 488 Phalloidin (1:20, #8878s, CST, Danvers, USA) added to the cells. The nuclei were stained with DAPI. Fluorescence images were obtained using immunofluorescence microscopy (Thermo Fisher Scientific, Bothell, WA, USA). The THP-1 cells were immunofluorescence stained with the same procedure.