F4 80

The clinical tissue specimens of renal fibrosis were obtained from The First Affiliated Hospital of Sun Yat-sen (Guangzhou, China). IHC staining was performed as previously described^{21 (link)}. Target proteins were detected with antibodies against NLRP3 (Servicebio, Wuhan, China), FIP1 (Abclonal, Wuhan, China), collagen I (Sevicebio, Wuhan, China), Fibronectin (Servicebio, Wuhan, China), CD3 (Servicebio, Wuhan, China) and F4/80 (Servicebio, Wuhan, China) and evaluated by two independent examiners who were blinded to the animal groups.

For immunofluorescence analysis, paraffin-embedded liver sections were incubated with primary antibodies against F4/80 (1:3000, Servicebio, China) and iNOS (1:200, Abcam, USA), followed by incubation with a secondary antibody (Servicebio, Wuhan, China). Apoptotic cells were evaluated using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay kit (Roche Applied Science) according to a standard protocol. Liver myeloperoxidase (MPO) activity was assessed using immunohistochemistry. The sections were incubated with primary antibodies against MPO (Servicebio, Wuhan, China), followed by the application of the appropriate secondary antibody (Servicebio, Wuhan, China). Five microscopic fields were examined for each section and used for calculations.

To observe CXCL1/CXCR2 expression and inflammatory infiltration, IHC staining was performed on 4-μm-thick paraffin-embedded kidney sections with a Servicebio immunohistochemical kit (G1212-200T). After deparaffinization, rehydration, antigen repair, and blocking, paraffin sections of renal tissues were incubated with CXCL1 (Proteintech, 1:200), CXCR2 (Proteintech, 1:100), and F4/80 (Servicebio, 1:1,000) for 12 h in 4°C, The tissues were incubated with HRP-conjugated secondary antibody (Abcam, 1:200) for 30min at 37°C. Then, a DAB kit was used for development, followed by observation under a light microscope.

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.

Multiplex immunofluorescence (mIF) staining was performed using Opal 7-Color fIHC Kit (PerkinElmer, Waltham, United States) according to protocols which have been described previously (Parra et al., 2017 (link); Parra et al., 2018 (link)). The embedded tumor tissues that underwent deparaffinization and rehydration were heated at 95°C for 20 min using Tris–EDTA buffer or citrate buffer to retrieve antigen. Next, the slides were incubated with primary antibodies overnight at 4°C. Then, the slides were washed three times with 2-methyl-2H-isothiazol-3-one and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 10 min at room temperature. Next, the slides were incubated at room temperature for 10 min with one of the following Alexa Fluor tyramides included in the Opal 7 kit to detect antibody staining. After BOND Wash Solution washing, the slides were counterstained with DAPI for 5 min to visualize nuclei and then mounted with glycerine. The slides were scanned using the Pannoramic MIDI System (3DHISTECH, Budapest, Hungary). The following primary antibodies are used in this study: CD8 (1:50, Servicebio, Wuhan, China), Ly-6G (1:200, Servicebio, Wuhan, China), and F4/80 (1:200, Servicebio, Wuhan, China).

Pancreas tissues were fixed with 4% paraformaldehyde and then embedded in paraffin and cut into slices. For immunohistochemical analysis, a portion of paraffin sections were routinely stained with hematoxylin and eosin (H&E), while others were incubated with antibodies against Ki-67 (Signalway Antibody, USA), Insulin (Proteintech, USA), and F4/80 (Cell Signaling Technology, USA). Islets from consecutive tissue cross-sections were photographed at identical exposure conditions and magnification (400×) using a microscope (Leica). For immunofluorescence analysis, paraffin slides of 3-4 μm were prepared, and then incubated with rabbit anti-mouse F4/80 and rabbit anti-mouse iNOS (Servicebio, China). FITC (green) and Cy3 (red)-conjugated goat anti-rabbit IgG (Servicebio) were used to visualize F4/80 and iNOS, respectively. DAPI (4′, 6-diamidino-2-phenylindole) was used to stain the cell nuclei (blue). Images were captured with a fluorescence microscope (Leica). Detailed characteristics of the antibodies used are presented in Table S4.

Liver tissues were fixed with formalin and then embedded in paraffin and sectioned into a thickness of 4 μm. Hematoxylin and eosin (HE) staining was performed in order to determine the extent of liver necrosis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed on tissue sections according to the manufacturer’s instructions (In situ cell death detection kit, TMR red, Roche, Basel, Switzerland). To examine macrophages and neutrophils infiltration, paraffin-embedded sections were immunohistochemically stained with Ly-6G (Servicebio, GB11229) and F4/80 (Servicebio, GB11027) antibodies.