T8200

Macrophages were washed with PBS three times. Four percent fixative solution (Solarbio, P1110) was added to the Petri dish for 10 min. Then, the solution was removed and cells washed three times. Next, 0.5% Triton X-100 (Solarbio, T8200) was added to the dish for 10 min. The solution was removed and cells washed three times. Five percent BSA (Solarbio, A8010) was added to the dish, and it was incubated for 1 h at room temperature. Then, primary antibodies were added to the M0 (GLRX1: 1:500, Abcam, ab45953; CD11b: 1:100, Proteintech, 66519-1-lg) and M2 macrophages (GLRX1: 1:500, Abcam, ab45953; CD163: 1:100, Abcam, ab156769) (18 (link)), respectively, and they were incubated overnight at 4°C. The solution was removed and cells washed three times. Secondary antibodies (DyLight 488 goat antirabbit polyclonal antibody, Abcam, ab96899, 1:200; DyLight 594 goat antimouse polyclonal antibody, Abcam, ab96881, 1:200) were used for 1 h at room temperature. The solution was removed and cells washed three times. Prolong™ Diamond Antifade Mountant with DAPI (Invitrogen, P36962) was added to the dish, and photos were taken with confocal microscopy.

The paraffin-embedded kidney tissues were cut into 3-μm sections, dewaxed, rehydrated, and antigen retrieved as previously described. To increase the cell membrane permeability to antibodies, 0.2% Triton 100X (Solarbio, T8200) was extracted. After incubation with the blocking serum for 1 hour at room temperature (approximately 25°C), the sections were stained with the anti-LC3B antibody (ab51520, Abcam, 1 : 1000) and the anti-p62 antibody (ab56416, Abcam, 1 : 200) at 4°C overnight, followed by the donkey anti-rabbit/mouse IgG (H + L) highly crossadsorbed secondary antibody (A-21206/A-21203, Invitrogen, 1 : 1000). The nuclei were counterstained with 4′6-diamidino-2-phenylindole. Image acquisition and processing were performed as previously described. The immunofluorescence staining was assessed with a semiquantitative scoring method by two independent observers unware of the treatment received by each group.

Cells were cultured in 24-well plate. Following treatments, samples were fixed in 4% paraformaldehyde,permeabilized in 0.5% Triton X-100 (T8200, Solarbio, Beijing, China)and blocked with 5% goat serum, followed by incubation with anti-COL2A (1:100, YT1022, Immunoway), anti-GSDMD (1:100, A18281, ABclonal) and anti-GRP78 (1:200, ab21685, Abcam) overnight at 4°C. Subsequently, samples were incubated with secondary antibodies (Invitrogen, United States) for 1 h in the dark. Cell nuclei staining was performed with DAPI (Solarbio, China) and photographed immediately with a fluorescence microscope (IX73, Olympus, Japan).

Expanded islet clusters were harvested using cell recovery solution (Corning, 354253) and fixed in 4% paraformaldehyde for 30 min at room temperature, followed with blocking and permeabilizing in PBS with 0.5% Triton X-100 (Solarbio, T8200) and 5% donkey serum (Solarbio, SL050) for 30 min at room temperature. Then, samples were incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody for 2 h at room temperature. DAPI (Beyotime, C1002) was used to stain the nucleus and find islets. The following antibodies were used for immunofluorescence: anti-insulin (1:200, sc-9168; Santa), anti-somatostatin (1:600, ab30788; Abcam), anti-glucagon (1:200, G2654; Sigma). anti-PDX1 (1:200, ab47267; abcam), anti-SOX9 (1:200, ab185966; abcam), anti-NKX6.1 (1:200, ab221549; abcam), anti-MAFA (1:200, ab26405; abcam), anti-KI67 (1:200, D3B5; Cell Signaling Technology). Imaging of the expanded islet clusters was performed on Zeiss LSM 780 and processed using ImageJ or Adobe illustrator software.