Newborn gammarids freshly extracted from the female brood pouch were gently dried on a paper, pooled in groups of five individuals, and weighed using an ultra-microbalance (Sartorius Ultra Micro Balance MSU2.7S000DM, readability 0.1 μg, repeatability ± 0.25 μg).
Lipid droplets in newborn individuals were visualized using a stock solution of Nile red staining (technical grade, Sigma-Aldrich) prepared with 10 mg of Nile red to 100 mL of acetone (for HPLC, Carbo-Elba). Just before use, the working solution was prepared by diluting the stock solution to 2 mg L-1 in synthetic water. Live individuals were then exposed to the Nile red working solution in the dark for 3 h at 14°C. Stained individuals were immobilized between the microscope slide and the cover slide, and images of the entire animal were taken at 5 × magnification for visualization of lipid droplets. Fluorescence images were obtained using a Leica DM 2500 with a L5 filter cuber (EX 480/40, EM 527/30) [28 (link)]. Twenty individuals per condition were analyzed.
Lipid droplets in newborn individuals were visualized using a stock solution of Nile red staining (technical grade, Sigma-Aldrich) prepared with 10 mg of Nile red to 100 mL of acetone (for HPLC, Carbo-Elba). Just before use, the working solution was prepared by diluting the stock solution to 2 mg L-1 in synthetic water. Live individuals were then exposed to the Nile red working solution in the dark for 3 h at 14°C. Stained individuals were immobilized between the microscope slide and the cover slide, and images of the entire animal were taken at 5 × magnification for visualization of lipid droplets. Fluorescence images were obtained using a Leica DM 2500 with a L5 filter cuber (EX 480/40, EM 527/30) [28 (link)]. Twenty individuals per condition were analyzed.
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