Zen microscopy software 2011

At the stage of 1 day of adulthood, synchronized gst-4::GFP, sod-3::GFP, daf-16::GFP and skn-1::GFP transgenic worms fed heat killed OP50 and 10 mM 2-HIBA from embryo hatching were anesthetized with sodium azide (20 mmol L⁻¹) (Sigma-Aldrich, St. Louis, MO, United States) and observed by Zeiss Axiovert 25 microscope as described in (Bianchi et al., 2020 (link)). The experiments were repeated three times and 15 worms per group were used in each experiment. Images were taken at the time of exposure of 0.2 s and fluorescence was analyzed using ImageJ software. Scale bars were inserted by Zeiss ZEN Microscopy Software 2011.

About 10 worms of each condition were analyzed at the early stage (from day 1 to day 4) and during aging (from day 9 to day 11) of adulthood. Pumping rate was measured under Zeiss Axiovert 25 microscope by counting the number of grinder contractions of 10 animals for each treatment, during a period of 30 s, as described in (Guantario et al., 2018 (link)). To analyze the auto-fluorescence of lipofuscin granules, 10 worms per condition at different stages (day 2, 3, 5 and 11) were washed three times with M9 buffer and observed by Zeiss Axiovert 25 microscope under DAPI filter. Images were taken at the time of exposure of 0.2 s and scale bars were inserted by Zeiss ZEN Microscopy Software 2011. Fluorescence was analyzed using ImageJ software. Aging analyses were performed in triplicate.