Microscopic Imaging of C. elegans Larvae

For microscopy, iL3s, iL3as, and non-activated iL3s were screened for YC3.60 expression, mScarlet-I expression, and/or for the presence of Alexa Fluor on a Leica M165 FC fluorescence microscope following nicotine paralysis [32 (link)]. Animals were collected in a watch glass with BU saline. Animals were then mounted in droplets on a slide with a 5% Noble agar (dissolved in BU saline) pad, exposed to 100 mM levamisole (dissolved in BU saline), and covered with a cover slip. Epifluorescence and DIC imaging were performed using an inverted Zeiss AxioObserver A2 microscope equipped with a Plan-APOCHROMAT 20X objective lens, a Colibri 7 (Zeiss) for LED fluorescence illumination, a 38 HE filter set for GFP (BP470/40, FT495, BP 525/50), a 63 HE filter set for mScarlet-I or Alexa Fluor (BP572/25, FT590, BP629/62), a Hamamatsu ORCA-Flash4.0 camera, and Zen 3.3 (blue edition) software (Zeiss). Images were captured as z-stacks and maximal intensity projections were constructed using Fiji [68 (link)].