Fv3000 confocal microscopy

Cells grown to subconfluence were fixed with cold methanol for 10 min, permeabilized with 0.1% Triton X-100 in PBS, blocked with 5% BSA, and incubated overnight at 4 °C with the primary antibodies in PBS with 5% BSA. Cells were then washed twice in PBS and incubated with secondary antibodies at room temperature for 1 h. Cells were mounted in Fluoromount-GTM with DAPI (Invitrogen) and images were acquired using Olympus FV3000 Confocal Microscopy with an UPlanXApo 60X/1.42NA objective. The Drosophila fat bodies were dissected from second instar larvae, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.3% Triton X-100 in PBS, and blocked with 5% normal goat serum in 0.1% Triton-PBS. Tissues were incubated overnight with the primary antibodies in PBS with 5% goat serum, and 0.3% Triton X-100. After washing twice in PBS and incubating with secondary antibodies, tissues were stained with DAPI and mounted in 90% glycerol in PBS. For LysoTracker staining, larvae fat body were dissected in PBS and immediately incubated in PBS with 1 μM LysoTracker Red DND-99 (Invitrogen) and DAPI. Samples were washed in PBS and mounted on slides. Images were acquired with a confocal laser scanning microscope (Olympus FV3000).

HEK293T cells or hippocampal neurons grown on coverslips were washed with PBS and fixed at room temperature using 4% paraformaldehyde (PFA) in PBS for 10 min or 4% PFA and 4% sucrose in PBS for 15 min, respectively. For immunocytochemistry of endogenous PSD-95, hippocampal neurons were incubated with blocking solution (2% normal goat serum (NGS), 0.2% Triton X-100, PBS) for 1 h at room temperature. Cells were washed with PBS twice and then incubated in antibody solution (2% NGS, 0.2% Triton X-100, PBS) with anti-PSD-95 antibody (Millipore, MABN68) for overnight at 4°C. Cells were washed with PBS 3 times and incubated with antibody solution with Alexa Fluor 568-conjugated anti-mouse IgG antibody (Molecular Probes, A-11019) for 1 h at room temperature. Cells were washed with PBS 4 times and embedded with Mounting Medium (Vectashield, H-1000). Cells were observed using FV3000 confocal microscopy (Olympus) equipped with a 40× objective lens (Olympus, N2246700), a 60× objective lens (Olympus, N1480700), or a 100× objective lens (Olympus, N5203100). Immersion oil (Olympus, IMMOIL-F30CC) or silicone immersion oil (Olympus, SIL300CS-30CC) were used for 60× and 100× lenses, respectively.

MitoTracker^® Deep Red FM is used for mitochondrial localization. After cells culture, the medium was discarded, and a 500 nM MitoTracker^® Deep Red FM working solution (Invitrogen, Carlsbad, CA, USA) was added and incubated for 20 min at 37 °C. Then washed and fixed with 4% paraformaldehyde, the cells were stained with Hoechst 33,342 (Beyotime, Shanghai, China). Finally, FV3000 confocal microscopy (Olympus, Japan) was used for observation.

Immunostaining was performed by a general protocol. Cells were fixed with 4% PFA (paraformaldehyde) at RT for 10 min, permeabilized with 0.3% Triton X-100 at RT for 10 min, blocked in blocking buffer (PBS containing 5% goat serum, 1% BSA and 0.3% Tween-20) for 60 min, incubated with the 1st antibody in blocking buffer at RT for 2 h or at 4 °C overnight, and finally counter-stained with 1 μg/ml Hoechst 33342 (BBI, E607328). Pax7 (Bioss, bs-22741R, dilution 1:200), MyoD1 (Abcam, ab209976, dilution 1:200) antibodies for PSCs/myoblast, Desmin (Bioss, bs-1026R, dilution 1:200) for myofiber/myotube, and HoxC9 (Bioss, bs-7982R) for adipocytes were used as 1st antibodies. Goat anti-rabbit IgG secondary antibody, Alexa Fluor 488 (Beyotime, A0423, polyclonal, dilution 1:500) were used as 2nd antibody. All fluorescence images were taken by Olympus FV3000 confocal microscopy.
Nuclei counts were determined by quantifying Hoechst 33342 staining using ImageJ, and normalized against respective controls. The fusion index was determined by quantifying nuclei within desmin-stained myofibres as a proportion of total nuclei and multiplying by 100.
To visualize lipid droplet in adipocytes, after fixation with 4% PFA at RT for 30 min, 3 times PBS washing for 5 min, and soak in 60% isopropyl alcohol for 2 min, Oil red O and hematoxylin staining were performed successively.