Cm1510

Serial 10‐μm sections were made with a cryostat (CM1510; Leica, Tokyo, Japan) at −20°C and mounted on polylysine‐coated slides. Whole sections were stained for hematoxylin and eosin, succinate dehydrogenase (SDH), fast myosin heavy chain (MHC), and Oil Red O. SDH activities in individual muscle fibers of the histological sections were examined and analyzed as described previously (Eshima et al. 2013, 2015). Mouse monoclonal antibodies that react specifically with the type IIa (1:1,000; SC‐71) and IIx (1:100; BF‐35) MHC isoforms were supplied by Developmental Studies Hybridoma Bank (University of Iowa, IA). The M.O.M. Immunodetection kit (Vector Laboratories, Burlingame, CA) and Vectastain ABC kit (Vector Laboratories) were used to reveal the immunohistochemical reaction, according to the manufacturer's instructions. Intracellular myocellular lipid (IMCL) levels were assessed by Oil Red O staining (Koopman et al. 2001; Shortreed et al. 2009). The cross‐sectional areas, SDH activities, and IMCL levels were measured by tracing fiber outlines of approximately 169 fibers per section from muscle sections. The images were digitized as gray‐level pictures. Each pixel was quantified as one of 256 gray levels and then automatically converted to an optical density using ImageJ software.

Cervical dislocation and tissue harvesting were performed at various timepoints following behavioral assessments. Small and large intestines were flushed with PBS and optimal cutting temperature (OCT) compound (Tissue-Tek), and fresh-frozen in liquid nitrogen. Tissue was thawed to −20°C and subsequently sectioned onto glass slides using a cryostat microtome (CM1510, Leica, Germany) for immunofluorescence analysis.

The dermal safety of the tested IL/O MEs was investigated on YMP skin according to a previous report [9 (link)]. First, the desired size (2 cm × 2 cm) skins were treated with the IL/O MEs or D-PBS (1.0 mL) for 24 h where D-PBS was used as the control. The skin samples were then immersed into Histo Prep compound (Fisher Scientific, Branchburg, NJ, USA) at −80 °C followed by sectioning using a cryostat microtome (CM1510; Leica, Wetzlar, Germany) and placed on glass slides. The slides were then stained with hematoxylin and eosin solution (Muto Pure Chemicals Co. Ltd, Tokyo, Japan). Finally, the specimens were investigated under a high-powered light microscope (BZ-9000 BIOREVO, Keyence Corp., Itasca, IL, USA).

Hand-made gauze patches (approximately 0.5 × 1.0 cm²) immersed in PBS solution or S/O nanodispersions with FITC-PE or FITC-PE-GM were administered on the ear auricles of mice (1 mg/mL, 50 µg PE, or PE-GM/mouse). After 24 h, the patches were removed, the mice were sacrificed, and the ear auricles were harvested and washed with ethanol and PBS. The earpieces were embedded in optimal cutting temperature (O.C.T.) compound (Sakura Finetek, Tokyo, Japan) and frozen in liquid nitrogen. The frozen skin sections (20 μm) were prepared using a CM1510 cryostat microtome (Leica, Wetzlar, Germany), and imaged on a fluorescence microscope BZ-9000 (Keyence, Osaka, Japan).

For immunofluorescence analysis, hearts were harvested 3 days after arterial ligation and were immediately embedded in OCT and frozen at − 80 °C. Frozen samples were cut with a cryotome (Leica CM1510, Nussloch, Germany) into 10 μm sections, fixed with 4% formaldehyde, and blocked with goat serum. The sections were incubated with monoclonal PE-conjugated anti mouse CXCR4 (clone2B11, eBioscience) or respective PE-labeled isotype control (Rat IgG2b kappa, eBioscience) for 1 hour at room temperature. DNA was stained with 1 µg/mL DAPI (Sigma), and a coverslip was placed using mounting medium (DAKO). Primary antibodies were applied 1:100 (final dilution). Images were acquired using a LSM 880 confocal microscope with Airyscan module and Plan-Apochromat 20×/0.8 air objective (Carl Zeiss Microscopy) and processed using ZEN software (Zeiss).