Isolution dt 36

Tissues were fixed (10% formalin solution in 0.1 M PBS), paraffin embedded, and cut into 4-μm sections on a microtome. Tissue sections were stained with hematoxylin and eosin (H&E) under standard conditions. MIN6 cells were fixed in 2% paraformaldehyde for 40 min. Both the sections and cells were permeabilized in 0.1% Triton X-100 in PBS and blocked in 5% normal goat serum and 0.1% Triton X-100 in PBS prior to incubation with primary antibody. Islets were stained using Sirt6 Ab (Cell Signaling Technology, Beverly, MA, USA), Glut2 Ab (Santa Cruz Biotechnology, Dallas, TX, USA), glucagon Ab (Bioworld Technology, St Louis Park, MN, USA), and insulin Alexa Fluor® 488 (eBioscience, San Diego, CA, USA) together with DAPI nuclear stain (Invitrogen). Images were acquired using an Axiovert 40 CFL confocal microscope (Carl Zeiss, Oberkochen, Germany). Islet size was measured using iSolution DT 36 software (Carl Zeiss).

Bone marrow was isolated from the femurs and tibias of WT and KO mice and cultured in α-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS. Floating cells were defined as bone marrow macrophages (BMMs). To prepare conditioned medium (CM), BMMs (2 × 10⁶) were grown in α-MEM supplemented with 10% FBS. Confluent cells were treated with TNF-α (10 ng/ml), IL-1β (10 ng/ml), and IL-6 (10 ng/ml) for 3 h; washed 3 times; and cultured for a further 24 h; then, the supernatants were collected and used. Cell migration was assessed by determining the ability of the cells to move into a cell-free area in a two-dimensional scratch assay. Briefly, HaCaT cells (2 × 10⁶ cells) or MDFB cells (2 × 10⁶ cells) were grown in a 12-well plate. When cell confluence reached 90% or higher, fresh medium containing 10 μg/ml mitomycin C was added for 2 h. The cells in the center of the well were scratched with a 100-μl sterile pipette tip to create a cell-free area. The medium was changed to WT or KO BMM-derived CM. The scratched area was photographed using a microscopy system (Carl Zeiss) soon after scratching and 12 and 36 h later. The scratch area was measured using iSolution DT 36 software (Carl Zeiss).

Fixed liver tissues were embedded in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) for light microscopy. TUNEL staining was performed with commercial kits (Promega, Madison, WI, USA). Five to six random sections were investigated per slide to determine the necrotic area and percentage of apoptotic cells. To measure the necrotic area, sections were observed under an Axiovert 40 CFL microscope (Carl Zeiss, Oberkochen, Germany) and measured using iSolution DT 36 software (Carl Zeiss). For immunohistochemistry, sections were immunostained with antibody against IGFBP-3 (Santa Cruz Biotechnology) or 4-hydroxynonenal (4-HNE, Abcam).

Rotator cuffs with a humeral head were immediately placed in 10% formalin solution and decalcified in 10% EDTA for 2 months. Tissue sections were collected from the midline of the supraspinatus. For histologic findings, paraffin-embedded tissue sections (5 μm) were stained with hematoxylin and eosin (H&E). Masson’s trichrome staining was performed with a commercial kit (Abcam, Cambridge, UK). Safranin O staining was performed using Fast Green and Safranin O. Immunohistochemistry was performed using a specific HRP/DAP detection IHC kit (Abcam). Images were acquired using a Leica DM750 microscope (Leica, Wetzlar, Germany). The area of interest to measure for histological analysis was the tendon-to-bone insertion (enthesis) site. The area was measured using the iSolution DT 36 software (Carl Zeiss, Oberkochen, Germany).

We performed bony biopsies of the resected femurs to evaluate the bone healing potential. The resected femurs were fixed in 10% neutral buffered formalin and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 10 days or in a rapid decalcifying solution (Calci-Clear Rapid, National Diagnostics, Atlanta, GA, USA) for 12–24 h. To evaluate the histologic changes, paraffin-embedded tissue sections were stained with hematoxylin and eosin (Sigma-Aldrich, St. Louis, MO, USA). The area of newly formed bone was measured using the iSolution DT36 software (Carl Zeiss, Oberkochen, Germany).

Adipose tissues were immediately placed in fixative (10% formalin) solution. Histological sections (7 μm) were cut from formalin-fixed paraffin-embedded tissue blocks. Tissue sections were stained with hematoxylin–eosin (H&E) under standard conditions. Immunohistochemical staining was performed using a biotin-free immunoenzymatic antigen-detection system (Abcam, Cambridge, UK). For immunofluorescence staining, the sections were incubated with a combination of anti-perilipin (Fitzgerald, MA, USA) and anti-F4/80 (Abcam) at 4 °C overnight. After incubation with the corresponding fluorochrome-conjugated secondary antibodies, the sections were mounted and visualized using an LSM510 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany). The adipocyte area in the selected fat tissue sections was measured using iSolution DT 36 software (Carl Zeiss).