Imaging Lysosomal Markers in C. elegans

The epidermal lysosomal marker (P_CED-1::nuc-1::mCherry)^{43 (link)}, the pan-neuronal marker (P_RGEF-1::DsRed)^{54 (link)}, and the PVD/AQR-neuronal marker (P_F49H12.4::GFP from NC1687) were imaged after worms were mounted on Noble agar pads in M9 with NaN₃ (0.25%). Zeiss AxioImager M2 with a black and white Hamamatsu ORCA C13440 camera was used for DIC images. All images were taken at the same magnification for the same amount of time for comparisons as indicated in the relevant figure legends. Fiji (Image J) was used for lysosome quantification. The same threshold was applied to all mutants. For lysosomal structure, puncta with circularity of 0.4 to 1 were quantified as particles and puncta with circularity of 0–0.4 were quantified as tubules.

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Yuan W., Weaver Y.M., Earnest S., Taylor CA I.V., Cobb M.H, & Weaver B.P. (2023). Modulating p38 MAPK signaling by proteostasis mechanisms supports tissue integrity during growth and aging. Nature Communications, 14, 4543.

Publication 2023

AxioImager M2 ORCA C13440

Agar Epidermal Lysosomal Nan3 Neuronal Orca Worms

Corresponding Organization :

Other organizations : The University of Texas Southwestern Medical Center

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Variable analysis

independent variables

Genotype/Mutant

dependent variables

Lysosomal quantification and structure (puncta and tubules)

control variables

Mounting method (Noble agar pads in M9 with NaN3 (0.25%))
Imaging equipment (Zeiss AxioImager M2 with a black and white Hamamatsu ORCA C13440 camera)
Imaging conditions (same magnification, same exposure time)

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