Gb11027

Tissue was fixed overnight in neutral buffer formalin (HT501128, Sigma, St Louis, MI, USA), paraffin embedded, 5 μm thick and stained with hematoxylin and eosin (C0105S, Beyotime Biotech, Shanghai, China). The scale bars for the images shown in figure represents 200 μm. Immunohistochemistry was performed using standard protocols on deparaffinized sections using anti-F4/80 antibody (gb11027, Servicebio, Wuhan, China). Photographs were taken using an optical microscope (Ci-L, Nikon, Tokyo, Japan). Adipocyte sizes were analyzed by Image J. Briefly, at 100× magnification, five random fields of each slide were captured. Immunohistochemical analysis was performed using ImageJ (National Institutes of Health, Bethesda, ML, USA).
The histochemical score of colitis was obtained by summing the following two parts: (a) Epithelial structure score: normal = 0, cup shape cell loss = 1, goblet cell loss = 2, crypt loss = 3, crypt mass loss = 4; (b) Immune cell infiltration score: no infiltration = 0, infiltration around the crypt = 1, mucosal muscular infiltration = 2, mucosal muscular infiltration with mucosal thickening and massive edema = 3, submucosal infiltration = 4.

Human kidney biopsy tissues of DN patients (n = 8) and glomerular minor lesion (GML) patients (n = 5) were recruited. GML is identified as minor morphological lesions by renal biopsy without DM. The percentage of tubular atrophy and interstitial fibrosis (IFTA) were assessed by two pathologists. The Ethics Committee of the Second Xiangya Hospital approved the protocol for this study. All patients signed approved informed consent forms. Renal tissues from mice were routinely processed, embedded in paraffin, sectioned (3–4 μm) and then subjected to HE staining.
Renal sections from human and mice were deparaffinized and rehydrated before being subjected to antigen retrieval in a microwave oven. Immunohistochemistry (IHC) was performed using anti-ALPK1 (1:200, Immunoway), CD68 (ZM-0060, ZSGB-Bio), F4/80 (GB11027, Servicebio), α-SMA (ab5694, abcam) and FN (ab2413, abcam) antibody as primary antibodies followed by secondary antibodies for2h. Then the slides were developed using a DAB detection kit. The human and mouse tissue sections were examined by light microscopy. Image J software (National Institutes of Health, Bethesda, MD) was used to measure the average intensity of at least 20 randomly selected fields.

Plasma insulin and hepatic triglyceride and cholesterol were measured using commercial kits. Glucose tolerance test (GTT), insulin tolerance test (ITT) and histological analyses, including hematoxylin & eosin (H&E) and Alcian blue-periodic acid Schiff (AB-PAS) staining, immunohischemistry of TLR4 (ab13867, Abcam), F4/80 (GB11027, Servicebio), and UCP1 (ab155117, Abcam), immunofluorescence staining of LC3 (14600-1-AP, Proteintech), were performed as described previously.^{52 (link)} Adipocyte size was determined from H&E stained sections and the number of CLSs was manually counted from F4/80-stained sections in five random fields from 5 mice per group by Image J software. Intestinal permeability was assessed in vivo following oral administration of fluorescein-isothiocyanate (FITC)-dextran (46944–500 MG-F, Sigma). Fecal SCFAs levels were detected by GC/MS (Thermo, TRACE 1310-ISQ LT), and the final data were normalized according to the fecal weight.

TMJ samples were collected, using 4% paraformaldehyde and 10% EDTA for fixation and decalcification, and following embedded in paraffin, continuous mid‐sagittal sections of 4 μm were cut for subsequent staining. After dewaxing and gradient hydration, haematoxylin and eosin (HE) staining was performed to quantify the synovial inflammation.
Immunofluorescence staining was incubated with the designated primary antibody overnight at 4°C and then conjugated the primary antibody with a fluorescent secondary antibody to visualize staining, and 4′,6‐diamidino‐2‐phenylindole (DAPI) reagent was used to develop nuclei. Primary antibodies were as follows: rabbit anti‐ALPK1 (1:300, 19107‐1‐AP; Proteintech), mouse anti‐CD68 (1:500, 66,231‐2‐Ig, Proteintech), rabbit anti‐Vimentin (1:200, 10366‐1‐AP, Proteintech), rabbit anti F4/80 (1:200, GB11027, Servicebio) and rabbit anti‐INOS (1:500, GB11119, Servicebio).
Immunohistochemical staining was to incubate with the designated primary antibody at 4°C overnight, then with the designated secondary antibody for 30 min, and finally developed the colour with DAB (DAB‐0031, Bio technologies), followed by counterstaining of nuclei with haematoxylin. Primary antibodies were as follows: rabbit anti‐TNF‐α (1:400, 11948S, Cell Signaling Technology), rabbit anti‐IL‐1β (1:400, YT2322, Immunoway) and rabbit anti‐IL‐6 (1:400, A14687, ABclonal).

ZO-1 and F4/80 staining of the colon was detected by staining the colonic tissue sections (5 μm) with anti-ZO-1 antibody (Servicebio, GB11195, 1:200 dilution) in PBS, anti-F4/80 antibody (Servicebio, GB11027, 1:1000 dilution) in PBS and goat-anti-rabbit CY3 conjugated antibody (Servicebio, GB21303, 1:300 dilution) in PBS. Finally, the sections were counterstained with DAPI (Servicebio, G1012). At a temperature of − 18°C, 20 μm frozen brain sections were cut using a cryostat. The brain slices were blocked with 3% bovine serum albumin for 30 min at room temperature and then incubated with the primary antibodies at 4°C overnight. The primary antibody anti-Iba1 (Servicebio, GB13105, 1:500 dilution) and anti-GFAP (Servicebio, GB11096, 1:800 dilution) were used. After washing with PBS, the sections were incubated with the secondary antibodies at 37°C for 50 min. The secondary antibody goat-anti-rabbit Cy3 conjugated antibody (Servicebio, GB21303, 1:300 dilution) were used. Finally, the sections were counterstained with DAPI (Servicebio, G1012) and then imaged with microscope (Nikon Eclipse C1). Quantification of positively stained cells in the PFC and HIP regions were used ImageJ.

Colon tissues were formalin fixed overnight at room temperature and then embedded in paraffin. 5 µm sections were stained with hematoxylin and eosin (HE) for pathological analysis. The pathological score was assessed by the severity of inflammation, the degree of inflammatory involvement, and the degree of epithelial/crypt damage as previously reported.^{58 (link)} Calculate each parameter and sum to get the total score.
For immunohistochemistry, paraffin-embedded tissue sections were stained with anti-MUC2 antibody (GB14110, 1:1000, Servicebio, China), anti-ZO-1 antibody (GB111402, 1:1000, Servicebio, China) and visualized by DAB staining according to manufacturer’s instructions.
For immunofluorescence, paraffin-embedded tissues were stained with anti-F4/80 antibody (GB11027, 1:1000, Servicebio, China), anti-Il-10 antibody (GB11108, 1:1000, Servicebio, China) and anti-CD206 antibody (GB113497, 1:500, Servicebio, China). The slides were independently assessed by two researchers who were unaware of the nature of the samples.