Bx51 dsu microscope

To measure the pancreatic islet area, paraffin-embedded pancreas total sections (5 µm) were stained with H&E, and percentages were obtained using i-Solution (IMT i-Solution Inc., Vancouver, BC, Canada) in three fields (150 × 150 µm²) randomly selected from two continuous sections (n = 4 per group). To determine the NAFLD activity score, paraffin-embedded liver sections (5 µm) were deparaffinized, stained with H&E, and subjected to histopathological examination using a BX51 light microscope (Olympus, Tokyo, Japan). The NAFLD activity score was quantified as the sum of the scores for steatosis (0–3), lobular inflammation (0–2), and hepatocellular ballooning (0–2) [20 (link)]. Nile red (Sigma-Aldrich), a fluorescent lipid droplet dye, was applied to assess hepatic lipid accumulation in HFD-/STZ-induced diabetic model mice. Nile red (Sigma-Aldrich) was used to stain frozen liver sections (5 µm) for 10 min. After washing, the sections were counterstained with Mayer’s hematoxylin (Sigma-Aldrich) for 45 s. The fluorescence intensity of each section was measured at 594 nm using a BX51-DSU microscope (Olympus).

Deparaffinized liver sections or frozen brain sections were incubated overnight with antibodies against F4/80 (sc-71085, Santa Cruz), Ly6G (ab25377, Abcam), and TREM2 (sc-373828, Santa Cruz) at 4 °C. After washing with 0.1 M PBS, sections were incubated with Alexa Fluor 488- and 594-conjugated donkey secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA, USA). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI; 1:20,000, Invitrogen). The fluorescence intensity of the solution was measured using a BX51-DSU microscope (Olympus). For the measurement of the intensity of immunostained protein from liver or brain sections, 12–16 fields (120 × 120 µm² or 20 × 20 µm²) were randomly selected from each section (n = 4 per group) and measured with i-Solution (IMT i-Solution Inc.).

For double immunostaining, free-floating brain sections were incubated with mouse anti-glial fibrillary acidic protein (GFAP, 1:200, Sigma-Aldrich) and rabbit anti-ionized calcium-binding adaptor molecule-1 (IBA-1, 1:200, Wako Pure, Osaka, Japan) or rabbit anti-phospho-Drp1 (Ser 616, 1:200, Cell signaling, MA, USA) either mouse anti-neuronal nuclei (NeuN, 1:200, Millipore, MA, USA) or rabbit anti-parvalbumin (1:200, Abcam) at 4 °C overnight and washed; sections were then incubated with the donkey secondary antibodies conjugated with Alexa Fluor 488- and 594-conjugated (Invitrogen, Carlsbad, USA). The images of the sections were visualized under a BX51-DSU microscope (Olympus).

Free-floating brain tissue sections were incubated with primary antibodies (Table 1) overnight at 4 °C. After washing, the sections were incubated with Alexa Fluor 488-, 594- or 680-conjugated secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA, USA). Nuclei were counterstained with DAPI (Invitrogen). Fluorescence was visualized using a BX51-DSU microscope (Olympus), and digital images were captured.

TUNEL analyses were performed to measure the degree of apoptosis in tissue using an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s protocol. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Waltham, MA, USA). Fluorescence was visualized using a BX51-DSU microscope (Olympus), and digital images were captured. TUNEL-positive cells were counted in the CA3 region (200 × 200 µm) in three sections (n = 4 mice per group) using ImageJ software (Version 1.52a, NIH, Bethesda, MD, USA).

To determine whether two molecules were in sufficient proximity to interact, the Duolink^® in situ PLA (OLINK Bioscience, Uppsala, Sweden) was performed according to the manufacturer’s manuals. Anti-rabbit MINUS (first PLA probe) detected rabbit Ex-4 antibody, whereas anti-mouse PLUS (second PLA probe) detected mouse GLP-1R antibody (Supplementary Table S1). After performing the assay, sections were stained with DAPI (1:10,000, Invitrogen, Carlsbad, CA, USA), washed, and coverslipped using mounting medium. Images of sections were captured by BX51 DSU microscope (Olympus).

Sections of deparaffinized skeletal muscle were incubated with primary antibodies (Table S2) for immunostaining. After washing three times with 0.1 M PBS, the sections were incubated for 1 h at room temperature with Alexa Fluor 488– or 594–conjugated donkey secondary antibody (Invitrogen). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1:10,000; Invitrogen), and fluorescence images of the sections were captured using a BX51-DSU microscope (Olympus). Immunofluorescent intensity data for each protein were obtained from selected images. Six fields (50 × 50 μm²) were randomly selected on each section using iSolution software (IMT iSolution Inc.). Intensity measurements are represented as the percentage of the mean number of pixels versus the corresponding value at which the pixel of the respective intensity was present.