Fifty age-synchronized worms were imaged every three days (days 3, 6, 9, 12, 15, 18). Worms were plated on Fluorodeoxyuridine (FUdR) treated agar plates in order to maintain only adult worms throughout the imaging process (Mitchell et al. 1979). Neurons were visible using the GABAergic specific
unc-25::GFP marker strain. To keep the data consistent, images were taken of the GABAergic axons and dorsal commissures between the VD5 and DD5 cell bodies (Panel A, scale bar 10 microns). Fluorescent images were taken using the Zeiss Axioplan fluorescent microscope under 40x objective. To prepare slides for fluorescence microscopy, an agarose immobilization protocol was followed (Fang-Yen et al. 2009). Aging of the neurons was quantified by counting the number of axonal beads and branches present on the neural processes. Branched processes emanating from the cell body, neural axon or dorsal commissures were scored as aging phenotypes. As predicted, GABAergic neurons displayed phenotypic evidence of aging.