Gb11229

Tissues were fixed in 10% neutral buffered, formalin and paraffin embedded and standard IHC was performed using rabbit anti-CD3 pAb (1:150 dilution; GB13014; Servicebio). Serial sections were stained with rabbit anti-CD68 pAb (1:500 dilution; GB11067, Servicebio) and rabbit anti-LY6G pAb (1:500 dilution; GB11229, Servicebio) and visualized using peroxidase/DAB. Negative controls were included by incubation of the sections only with a secondary antibody. Quantification was performed by counting positive cells in 6 to 10 high-powered fields (magnification, ×200) in a blinded fashion.

In order to evaluate the infiltration of inflammatory cells in pancreas, we detected CD45, Ly6G and F4/80 by immunofluorescence. Briefly, Frozen slides are baked in a 37 °C oven for 10–20 min and fix in paraformaldehyde for 30 min. Afterward, the slides were heated in an autoclave within EDTA antigen retrieval buffer (pH 8.0) for antigen repairing, followed by 3% BSA to block non-specific binding. Slides were then incubated with primary antibody including CD45 (GB11066, Servicebio, China, 1:1000), Ly6G (GB11229, Servicebio, China, 1:500) and F4/80 (GB113373, Servicebio, China, 1:500) at 4 °C overnight, and with Cy3 conjugated secondary antibody (GB21303, Servicebio, China, 1:500) at room temperature for 50 min in dark condition. After DAPI counterstain in nucleus, the sections were observed and images collected under the fluorescence microscope. The mean fluorescence intensity (MFI) was analyzed by ImageJ (V1.8.0.112). Five visual fields were randomly selected on each slide for MFI analysis.

IHC staining was performed according to the commercial kits (PV-6001 and PV-6002, ZSGB-Bio, Beijing, China). Antigen retrieval of the paraffin sections was performed using the same protocol described for IF staining. Subsequently, sections were incubated with 0.3% H₂O₂ for 10 min to inactivate endogenous peroxidase activity and then washed with PBS. After being blocked with 5% goat serum for 15 min, slices were incubated overnight at 4 °C with the primary antibodies as follows: rabbit anti-NLRP3 antibody (ab214185, 1:200, Abcam), rabbit anti-cleaved-caspase 1 p20 (1:100, AF4005, Affinity), IL-1β (1:100, GTX74034, Gentex), rabbit anti-GSDMD (1:800, ab219800, Abcam), mouse anti-IBA-1 (1:300, GB12105, Servicebio), rabbit anti-Ly6G (1:500, GB11229, Servicebio), rabbit anti-CD68 (1:100, DF7518, Affinity). The sections were incubated with enzyme-conjugated goat anti-mouse IgG or goat anti-rabbit IgG polymer for 30 min. Finally, immunoreactivity was visualized using 3,3-diaminobenzidine (DAB, ZLI-9017, ZSGB-Bio) followed by restaining with hematoxylin. Images were captured by a light microscope (Leica, DM2500, Germany).

Tissues were fixed in 10% buffered formalin and embedded in paraffin. The tissue sections were obtained from lung tissue and stained with hematoxylin–eosin (H&E). For immunofluorescence staining, the sections were rehydrated and washed in PBS, pretreated for 1 h at room temperature with protein block solution (Dako, Carpinteria, CA). After incubation with CD68 (Servicebio, GB14043, 1:200) and LY-6G (Servicebio, GB11229, 1:200) overnight at 4 °C, sections were washed and incubated with fluorescence-labeled secondary antibodies. After the nucleus was stained with Hoechst (Invitrogen, USA), samples were examined under the microscope.

Liver tissue sections were stained with H&E staining. To detect neutrophils, macrophages and lymphocytes in the liver tissue, paraffin-embedded mouse liver sections were stained by antibodies against Ly6G (1:1000, GB11229, Servicebio, Wuhan, China), F4/80 (1:1000, GB11027, Servicebio, Wuhan, China), CD3 (1:1000, GB11014, Servicebio, Wuhan, China) and B220 (1:4000, GB11066, Servicebio, Wuhan, China). To detect NET formation in the liver tissue, the sections were incubated with anti-citH3 (1:200, ab5103, Abcam), anti-NE (1:50, sc-55549, Santa Cruz) and anti-MPO (1:200, AF3667, R&D). 4′,6-Diamidino-2-phenylindole (2 μg/ml, DAPI, Servicebio, Wuhan, China) was used to detect DNA. Finally, slides were visualized using an Olympus microscope (IX73, Tokyo, Japan).