A simple fixation for detection of endogenous fluorescent reporters in C. elegans that nearly eliminates intestinal autofluorescence.

Abstract

Gut granules are prominent cytoplasmic organelles that auto fluoresce when excited by much of the visible spectrum confounding fluorescence imaging of endogenous fluorescent reporters. We report a simple, chemical-free fixation method for C. elegans that preserves non-cytoplasmic GFP localization for months while nearly eliminating intestinal autofluorescence. To illustrate the utility and limitations of the method, we present representative images of live and heat-fixed worms expressing a variety of membrane and non-membrane localized GFP reporters expressed in diverse tissues, cells, and cellular organelles. We also describe our experience with various experimental parameters. The observed variability of non-membrane-anchored reporters suggests prudent adopters should empirically interrogate the signal fidelity of their specific reporters upon heat fixation.