Fixation and Expansion Microscopy of Egg Extract Spindles.

In vitro systems based on egg extracts have elucidated many aspects of spindle assembly. Still, numerous unknowns remain, particularly concerning the variation in spindle morphologies. The and egg extract systems, which recapitulate diverse spindle sizes and architectures, serve as ideal tools to investigate the regulation of spindle morphometrics. However, fully understanding spindle architectural differences is hindered by the spindle's size and high microtubule density. Indeed, classical fluorescence microscopy lacks the resolution to detail the organization of spindle microtubules, and although electron tomography can distinguish individual microtubules, segmenting thousands of microtubules and tracking them across dozens of sections remains an unachieved challenge. Therefore, we set out to apply expansion microscopy to the study of egg extract spindles. During this process, we realized that optimizing spindle fixation as well was crucial to preserve microtubule integrity. Here, we present an optimized fixation and expansion microscopy protocol that enables the study of spindle architecture in egg extracts of both and . Our method retains the fluorescence of rhodamine tubulins added to the extracts and allows for both pre- and post-expansion immunofluorescence analysis. Key features • Expansion of optimally fixed spindle assembled from egg extracts of , and possibly others. • Retains the fluorescence of the rhodamine-tubulin that copolymerizes with endogenous tubulin within spindle microtubules, allowing their imaging without immunolabeling. • Compatible with both pre- and post-expansion immunolabeling to increase labeling possibilities. • Optimized spindle fixation that best preserves microtubule integrity for expansion and can also be used without expansion for regular immunofluorescence experiments.