Ecdysteroid sigma

Nile red determination . The Nile red stock solution was prepared in acetone and stored protected from light following Tingaud-Sequeira et al. (2011) . Just before use, the working solution was prepared by diluting the stock solution to μM in ASTM. Live individuals were then exposed to Nile red working solution in the dark for 1 hr at 20°C. After incubation, animals were placed in 100 mL ASTM for 1 min to allow clearance of Nile red residuals. Following clearance, animals were placed individually in -mL centrifuge tubes, the remaining water was removed, and samples were sonicated in 300 μL of isopropanol. The homogenized extract was then centrifuged at 10,000 × g . We used 200 μL of supernatant to measure Nile red fluorescence using an excitation/emission wavelength of 530/590 nm and a microplate fluorescence reader (Synergy 2, BioTek). Each treatment had one animal per sample (10 replicates in total). For each quantification and treatment, 10 blanks (animals not exposed to Nile red) were used to account for background levels of fluorescence. After exposure to Nile red, images were taken in the area surrounding the midgut for visualization of lipid droplets. Fluorescence and bright file images were obtained using a Nikon SMZ1500 microscope and a Nikon Intensilight C-HGFI with a GFP filter (EX 472/30, EM 520/35; Nikon).

Ecdysteroid sigma

ecdysteroid sigma

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ecdysteroid sigma