Mpo antibody

The lung tissue slides embedded in paraffin were exposed to citrate solution for antigen retrieval after dewaxing and rehydration. The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 15 min and incubated with MPO antibody (1:100 dilution; ABclonal, Shanghai, China) or P65 antibody (1:100 dilution; ABclonal, Shanghai, China) overnight at 4 °C. For double immunofluorescence staining, the slides were probed with PTPRO (1:50 dilution, Santa, USA) and keratin 19 (1:100 dilution; ABclonal, Shanghai, China) or beta IV Tublin (1:100 dilution; Affinity, Cincinnati, OH, USA). Then the tissues were incubated in the dark with a Cy3-conjugated goat anti-rabbit IgG (1:200 dilution; Beyotime, Shanghai, China) or FITC-labeled goat anti-mouse IgG (1:200 dilution; Abcam, Cambridge, UK) at room temperature for 1 h. The tissues were re-stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China), and the images were captured at 400 × magnification by a fluorescence microscope (BX53, Olympus, Tokyo, Japan).

Caspase-3, Caspase-8, RIPK1, RIPK3 (rabbit antibody), MLKL, OGT and OGA antibodies were purchased from Proteintech (Wuhan, China). Phospho-RIPK1, phospho-RIPK3, phospho-MLKL and secondary antibodies were from Cell Signaling Technology (Beverly, MA, USA). MPO antibody was provided by ABclonal Technology (Wuhan, China). CD68 antibody was from Abcam (Cambridge, MA). O-GlcNAc antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Another RIPK3 (mouse) antibody was purchased from Novus Biologicals (Littleton, CO, USA). 2,4,6-trinitrobenzene sulfonic acid (TNBS) was from Thermo Fisher Scientific (CA, USA). Protein A/G magnetic beads were purchased form Bimake (Shanghai, China). The TUNEL staining and immunohistochemistry kits were purchased from Wuhan Gugeshengwu Technology Co.,Ltd. (Wuhan, China). All other regular reagents were from Wuhan Gugeshengwu Technology Co.,Ltd. unless otherwise specified.