F4 80 antibody

Lipopolysaccharides (Escherichia coli O111:B4, L2630, for animal experiments; Escherichia coli O111:B4, L4391, for cell experiments) were purchased from Merck (New Jersey, USA). ACSS2 inhibitor (cat no: S8588) was purchased from Selleck (Houston, USA). ML264 (cat no: HY-19994) was purchased from MedChemExpress (New Jersey, USA). Lotus Tetragonolobus Lectin (LTL) (cat no: FL-1321-2) was purchased from Thermo Fisher (MA, USA).
The following antibodies were used. ACSS2 antibody (cat no: ab133664) from Abcam (Cambridge, UK); KIM-1 antibody (cat no: sc-518008) from Santa Cruz (California, USA); F4/80 antibody (cat no: 28463-1-AP) from Proteintech (Wuhan, China); Cleaved GSDMD (N Terminal) antibody (cat no: A22523) and NLRP3 antibody (cat no: A5652) from Abclonal (Wuhan, China); caspase-1 antibody (cat no: 24232), anti-IL-1β antibody (cat no: 12242) from Cell Signaling Technology (Massachusetts, USA); GSDMD antibody (cat no: AF4012), NF-κB p65 antibody (cat no: AF5006), phospho-NF-κB p65 (Ser536) antibody (cat no: AF2006), and KLF5 antibody (cat no: AF7542) from Affinity (Changzhou, China).

Berberine chloride dihydrate (Berberine hydrochloride, purity: 98%) was obtained from Yuanye Inc. (Shanghai, China) and Ibuprofen (purity: 98%) was obtained from Aladdin (Shanghai, China). Both of them were used directly after purchase. All other solvents were analytically pure. Deionized water was used in this study. All plasmids were supplied by Shanghai GenePharma Co. Ltd (Shanghai, China). Sources of antibodies were as follows: IKKε antibody (Cell SignalingTechnology, 2690), TBK1 antibody (Cell Signaling Technology, 3013), Phospho-TBK1 Ser172 antibody (Cell Signaling Technology, 5483), Phospho-AMPKα Thr172 antibody (Cell Signaling Technology, 2535), Anti-UCP1 antibody (Abcam, ab10983), F4/80 antibody (proteintech, 28463-1-AP), GAPDH antibody (proteintech, 60004-1-Ig), Anti-rabbit IgG (Cell Signaling Technology, 7074 S), Anti-mouse IgG (Cell Signaling Technology, 7076 S), CL488-conjugated Affinipure Goat Anti-Mouse lgG (H + L) (proteintech, SA00013-1), CL488-conjugated Affinipure Goat Anti-Rabbit lgG (H + L) (proteintech, SA00013-2). 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, ISO and Oil Red O solution (0.5% in isopropanol) were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant Murine TNF-α was purchased from Pepro Tech (Rocky Hill, NJ, USA). The BCA protein assay kit was purchased from Beyotime Biotechnology Co., Ltd (Beijing, China).

Lung tissues were fixed with 4% paraformaldehyde solution and embedded in paraffin. Then, 5 µm thick sections were deparaffinized in serial solutions of xylene, gradient ethanol, and water, followed by antigen retrieval by steaming in 1 mM EDTA for 5 min in a pressure cooker. The lung sections were washed with PBS buffer and blocked with 5% BSA for 30 min. Next, blocked samples were incubated with mouse anti-α-SMA (1:200; Servicebio, Wuhan, China) or anti-TREK–1 antibody (1:200, Alomone Labs, Jerusalem, Israel) and F4/80 antibody (1:200, proteintech, Wuhan, China) overnight at 4 °C followed by exposure to staining with corresponding secondary antibodies for 1 h at room temperature. Slides were then counterstained with nuclear dye DAPI and mounted. Images were acquired on a fluorescence microscopic imaging system (Leica, Weztlar, Germany).