Eclipse ti confocal system

For fat body/adipose immunostaining, carcass was dissected (with all eggs and intact intestines removed) in 1X PBS and fixed with 4% paraformaldehyde for 20 min at room temperature, washed 3 times with 1X PBS containing 0.2% Triton X-100 (1X PBST) and then blocked in blocking buffer (5% BSA in 1X PBST) for 1h. Primary antibodies; anti-ATP5A (ab14748, 1:500, Abcam) was applied overnight at 4 °C. Samples were then incubated with Alexa Flour-conjugated secondary antibodies (Jackson Immunoresearch, 1:500), and fresh Nile Red solution (1 μL 0.004% Nile Red Solution in 500 μL 1X PBS) with Hoechst (DAPI; 1:500), overnight at 4 °C, followed by rinsing with 1X PBS. Confocal images were collected using a Nikon Eclipse Ti confocal system (utilizing a single focal plane) and processed using the Nikon software and Adobe Photoshop. ATP5A Staining intensity was quantified using ImageJ. The image scale was set according the image size (Analyze > set scale). The ATP5A signal was marked using freehand selection tools. The area of marked signal was measured using the measure tool (Analyze > measure). At least 3–5 individual fat body whole images (using a global scale) were measured and averaged.

For muscle immunostaining, dorsal longitudinal thorax muscle segments were dissected in PBS and fixed with 4% paraformaldehyde for 20 min at room temperature, washed 3 times with PBS containing 0.2% Triton X-100 (PBST) and then block in blocking buffer (5% BSA in PBST) for 1 h. Primary antibodies; anti-ATP5A from Abcam (ab14748, 1:500) and anti-GDH from Sigma (Anti-GLUD1, HPA061369, 1:500) were applied overnight at 4°C. Alexa Flour-conjugated secondary (Jackson Immunoresearch, 1:500) antibodies, Alexa Fluor 555 Phalloidin (Thermo Fisher Scientific, 1:500) and Hoechst (DAPI; 1:500) were incubated overnight at 4°C.
Confocal images were collected using a Nikon Eclipse Ti confocal system (utilizing a single focal plane) and processed using the Nikon software and Adobe Photoshop.