Scmm r2

The in situ TG activity was visualized using FITC-labeled peptides from the unfixed kidney sections as reported previously^{21 (link)}. The frozen specimen block was cut into 10-μm-thick sections using cryomicrotome (Leica Microsystems). These sections were collected using cryofilm, and then incubated in a reaction mixture containing 100 mM Tris-HCl (pH 8.0), 1 mM DTT, and 5 mM CaCl₂ in the presence of 5 μM FITC-labeled peptide at 37 °C for 1 h. Following this, the sections were washed and mounted using SCMM-R2 (Leica Microsystems) or Fluoromount/Plus (Cosmo Bio). The fluorescence signals were observed under a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). The signal intensities in the images were adjusted to maintain linearity using imaging software (Adobe Photoshop CS). Each experiment was conducted in triplicate.

Four weeks after transplantation, the mice were killed by isoflurane. The gastrocnemius muscles were then removed from normal and diabetic mice and fixed with 4% paraformaldehyde overnight. To characterize the transplanted hDPSCs, frozen gastrocnemius muscle sections were observed by fluorescence microscopy. For this purpose, the muscles were embedded in a specific immersing solution (SCMM) in liquid nitrogen and cooled isopentane, and the frozen samples were cut serially into 5-μm-thick sections. The sections were subsequently mounted using adhesive film (Cryofilm type 1; Leica Microsystems, Wetzlar, Germany) and mounting medium (SCMM-R2; Leica Microsystems). The sections were then incubated with primary antibodies, including an anti-human nuclei antibody (Millipore). Human nuclei were detected using the Zenon Alexa Fluor 488 Mouse IgG1 Labeling Kit (Life Technology Co, Tokyo, Japan), and the sections were analyzed by fluorescence microscopy (Leica AF6000LX, Leica Microsystems).