Gb11224

Liver tissue and ileum were fixed in 4% paraformaldehyde at 4°C overnight, then embedded in paraffin wax or snap-frozen in liquid nitrogen and stored at −80°C. Paraffin sections were stained with hematoxylin-eosin (H&E) for pathological analysis. NALFD activity score (NAS) is calculated from the semi-quantitative evaluation of hepatic steatosis, lobular inflammation, and hepatocyte ballooning, as the previous review concluded (Aron-Wisnewsky et al., 2020 (link)). For immunohistochemistry, liver sections were incubated with antibodies against F4/80 (gb11027, Servicebio, China) and myeloperoxidase (MPO) (gb11224, Servicebio, China). The number of positive cells in the liver sections was normalized to the tissue area. Ileum sections were incubated with antibodies against zonula occludens 1 (ZO-1) (ab96587, Abcam, United Kingdom) and occludin (ab216327, Abcam, United Kingdom). Images were captured using an optical microscope (Olympus BX51, China).

Paraffin-embedded sections were deparaffinized and incubated with a 0.3% H₂O₂ solution to remove endogenous peroxidase activity. Antigen retrieval was performed with a citrate buffer (pH 6.8). Goat serum was used to block nonspecific antigens. Then, paraffin sections were probed with a rabbit anti-mouse myeloperoxidase (MPO) polyclonal antibody (dilution: 1/1000; GB11224, Servicebio, Wuhan, China) or with an anti-insulin mouse monoclonal antibody (dilution: 1/500; GB13121, Servicebio, Wuhan, China). The next day, sections were incubated with a goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody for 1 hour at room temperature. A diaminobenzene horseradish peroxidase color development kit was used for the color reaction. Finally, the sections were counterstained with hematoxylin and dehydrated. Sections stained only with the secondary antibody with the omission of the primary antibody served as negative controls. The staining-positive cells in the pancreas were counted using the ImageJ software. At least six visual fields of each mouse were studied. Data were expressed as MPO+ cells per microscopy field and the percentage of insulin+ cells in islets [13 (link)].

The following antibodies were used in this research:
For flow cytometry analysis: anti-rat-CD3 (#201406, BioLegend), anti-rat-CD4 (#201509, BioLegend), anti-rat-CD8a (#201712, BioLegend), anti-rat-CD11b/c (#201817, BioLegend), anti-rat-granulocytes (#550002, BD Pharmingen), and anti-rat-CD68 (MCA341A700, Bio-Rad).
For immunohistochemistry: anti-myeloperoxidase (MPO) rabbit antibody (GB11224, Servicebio, 1:1,000), anti-CD68 rabbit antibody (GB11067, Servicebio, 1:500), anti-CD19 rabbit antibody (GB11061-1, Servicebio, 1:400), anti-CD3 rabbit antibody (GB111337, Servicebio, 1:1,000), anti-rat-endothelial-cell-antibody-1 (RECA-1, ab9774, Abcam, 1:200), and anti-CXCL1 (ab86436, Abcam, 1:200).
For immunocytochemistry: RECA-1 (ab9774, Abcam, 1:400), anti-CXCL1 (ab86436, Abcam, 1:100), and secondary antibodies (goat anti-mouse IgG Alexa Fluor^® 488, 1:200; goat anti-rabbit IgG Alexa Fluor 594^®, 1:200; ZSGB-BIO).
For Western blot: anti-p-p38 (#4511, Cell Signaling Technology, 1:1,000), anti-p38 (#8690, Cell Signaling Technology, 1:1,000), anti-p-p65 (#3033, Cell Signaling Technology, 1:1,000), anti-p65 (#8242, Cell Signaling Technology, 1:1,000), anti-GAPDH (#5174, Cell Signaling Technology, 1:2,000), anti-p47 (PA5-104250, Thermo Fisher Scientific, 1:2,000), and anti-p-p47 (PA5-99359, Thermo Fisher Scientific, 1:1,000).

To assess inflammatory status, paraffin sections were incubated with a rabbit anti-MPO polyclonal antibody (dilution: 1/1000; GB11224, Servicebio, Wuhan, China) overnight. After washing, sections were probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody to produce a color reaction. Sections stained with a secondary antibody were used as negative controls. The results were analyzed using ImageJ software.