Microphot sa fluorescence microscope

After treatment with DHA, AA and BSA for 48 h, C2C12 cells were fixed in 4% formaldehyde for 10 min and then washed 3 times with PBS for 5 min each. The cells were permeabilized with 0.5% Triton X-100 for 10 min, followed with 3 washes with PBS for 5 min. Blocking was done for 1 h with blocking buffer consisting of 10% PBS, 1% BSA and 0.3% Triton X-100. Then, the cells were incubated with anti-myogenin Alexa488 green (1:300, 53-5643-82, eBioscience™ by Thermo Fisher, Schwerte, Germany) for one h at 4 °C in dark. After two PBS washes, cells were incubated with Hoechst 33342 (0.2 μg/mL, H1399, Invitrogen by Thermo fisher) for 5 min in dark. The cells were then washed once with PBS and once with water. Fluorescence imaging was obtained with CC-12 high-resolution color camera (OSIS) connected to Nikon Microphot SA fluorescence microscope (Nikon) and analyzed with CELL^F software. For each treatment multiple photos were taken and in each field. Myotubes number, number of nuclei incorporated in myotubes, and the total number of nuclei were scored. The differentiation index was calculated by counting the number of nuclei showing Alexa 488 staining divided by the total number of nuclei from the same field. The Fusion index was calculated by counting the number of nuclei in myotubes containing more than 2 nuclei divided by the total number of nuclei from the same field.

Different bovine tissues were cryo-sectioned (thickness liver: 10 μm; MLD: 12 μm; SCF 25 μm) using a Leica CM3050 S (Leica, Bensheim, Germany) cryostat microtome. Sections were air dried, fixed with 4% paraformaldehyde, and washed three times with PBS. Unspecific binding of the secondary antibody was blocked using 10 % goat serum in PBS for 15 min. Sections were incubated for 2 h at room temperature in a humidity chamber with the same primary antibodies as used for western blots, diluted 1:100 in PBS. Specific binding of primary antibody was detected with the respective goat anti rabbit IgG secondary antibody labeled with Alexa Fluor 488 (Molecular Probes, Eugene, USA). Nuclei were counterstained with 1 μg/ml Hoechst 33258 (Sigma-Aldrich, Munich, Germany). Slides were covered using ProLong Diamond Antifade Mountant (Thermo Scientific) and appropriate cover-slips. Negative controls were incubated either omitting the primary antibody or blocking the primary antibody with the respective peptide or recombinant protein. No unspecific binding of the secondary antibody, but some unspecific binding of the primary antibodies was detected. Immunofluorescence was visualized with a Nikon Microphot SA fluorescence microscope (Nikon, Duesseldorf, Germany) and an image analysis system equipped with CELL^∧F software and a CC-12 high resolution color camera (OSIS, Muenster, Germany).

Muscle (SM) was cryo-sectioned (8 µm thick) using a Leica CM3050 S cryostat microtome (Leica, Bensheim, Germany). Sections were air dried and fixed with 4% paraformaldehyde. After washing with phosphate buffered saline (PBS) and permeabilization with PBS-TritonX100 (PBST), unspecific binding of the secondary antibody was blocked using 10% goat serum in PBST for 15 min. Sections were incubated with the primary antibody against FNDC5-c-term (1∶100) for 1 h at room temperature in a humidity chamber. Specific binding of primary antibody was detected with the respective goat anti rabbit IgG secondary antibody labelled with MFP 488 (MoBiTec, Goettingen, Germany). Nuclei were counterstained with 1 µg/ml Hoechst 33258 (Sigma-Aldrich). Slides were covered using Mowiol mounting medium including 1,4-diazabicyclo[2.2.2]octan (DABCO, Carl Roth) and appropriate cover-slips. Negative controls were incubated either omitting the primary antibody or blocking the primary antibody with the respective peptide. No unspecific binding of the secondary antibody and only minimal unspecific binding of the primary antibody was detected. Immunofluorescence was visualized with a Nikon Microphot SA fluorescence microscope (Nikon, Duesseldorf, Germany) and an image analysis system equipped with CELL^∧F software and a CC-12 high resolution color camera (OSIS, Muenster, Germany).

Neutral lipid droplets were analyzed using Invitrogen's HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit for image-based high-content screening assays (Invitrogen, H34476, ThermoFisher Scientific, Waltham, Massachusetts). Staining was prepared following the protocol of the manufacturer. Briefly, VSMCs were fixed in formalin and incubated with HCS LipidTOX™ Red Neutral Lipid Stain. Plates were then sealed without washing and imaged. Images were captured on a Nikon Microphot-SA fluorescence microscope or Nikon Eclipse Confocal Microscope A1 Ti-E (images were recorded at 512 × 512 or 2,048 × 2,048 pixels with a pixel dwell of 3.1) using Nikon NIS-Elements software (Nikon Melville, NY). At least ten high-power fields per biological replicate were collected. Three independent experiments were performed.