Patel Ohm H, Weisman Alexandra S, Hunter Craig P
Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States.
MicroPubl Biol. 2025 Jul 18;2025. doi: 10.17912/micropub.biology.001673. eCollection 2025.
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 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.
肠道颗粒是突出的细胞质细胞器，当受到大部分可见光谱激发时会自发荧光，这会干扰内源性荧光报告基因的荧光成像。我们报告了一种简单的、无化学试剂的固定方法，该方法可将非细胞质绿色荧光蛋白（GFP）定位保存数月，同时几乎消除肠道自发荧光。为了说明该方法的实用性和局限性，我们展示了表达多种膜定位和非膜定位GFP报告基因的活虫和热固定虫的代表性图像，这些报告基因在不同组织、细胞和细胞器中表达。我们还描述了我们在各种实验参数方面的经验。非膜锚定报告基因观察到的变异性表明，谨慎的使用者在热固定后应凭经验询问其特定报告基因的信号保真度。
