Bz 9000 fluorescent microscope system

Each isolated pancreas was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Frozen sections were prepared as previously described.15 (link) Briefly, the sections were incubated at 4°C overnight with guinea pig anti-insulin antibody and either rabbit antiglucagon antibody or rabbit anti-Ki67 antibody (dilution, 1:500; Abcam, Tokyo, Japan). Afterward, the sections were washed with PBS, then further incubated for 1 h in a mixture of rhodamine-conjugated antiguinea pig IgG antibody, antirabbit IgG antibody, and DAPI (4′,6-diamidino-2-phenylindole) fluorescent stain solution (dilution, 1:2000; Dojindo Molecular Technologies, Inc, Tokyo, Japan).
For morphological analysis, five sections per pancreas were randomly selected at 150 μm intervals. The sections were analyzed for cell count and cell area using a BZ-9000 fluorescent microscope system (Keyence, Osaka, Japan).

The mice were killed in the fed state. The collected skeletal muscle was rapidly frozen in isopentane cooled with liquid nitrogen. Serial sections from the tibialis anterior muscles were cut 10‐μm thick using a Microtome Cryostat (Leica Biosystems, Wetzlar, Germany) and placed on MAS‐coated slide glass. Specimens were blocked with protein‐block serum‐free reagent (Dako X0909; Dako, Glostrup, Denmark) for 5 min at room temperature and incubated overnight at 4°C with primary antibodies with rat monoclonal immunoglobulin G1 for Laminin α‐2 (4H8‐2; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), skeletal muscle myosin heavy chain IIA (SC‐71; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) or skeletal muscle myosin heavy chain IIB (BF‐F3; Developmental Studies Hybridoma Bank), followed by 60‐min incubation in Alexa Fluor‐conjugated secondary antibody against goat anti‐rat immunoglobulin G1 (1:1,000; Alexa Fluor 594; Invitrogen, Waltham, MA, USA), goat anti‐mouse immunoglobulin G1 (1:500; Alexa Fluor 488; BioLegend, San Diego, CA, USA) and goat anti‐mouse immunoglobulin M (1:500; TRITC; SouthernBiotech, Birmingham, AL, USA) at room temperature. Images were captured using BZ‐9000 fluorescent microscope system (KEYENCE, Osaka, Japan). A cross‐sectional area (μm²) of muscle fibers and selected fields on muscle sections were measured using BZ2 analysis application (KEYENCE).