Accelerated protein retention expansion microscopy using microwave radiation

Protein retention expansion microscopy (ExM) retains fluorescent signals in fixed tissue and isotropically expands the tissue to allow nanoscale (<70 nm) resolution on diffraction-limited confocal microscopes. Despite the numerous advantages of ExM, the protocol is time-consuming. Here, we adapted an ExM protocol to vibratome-sectioned brain tissue of Xenopus laevis tadpoles and implemented a microwave (M/W)-assisted protocol (^M/WExM) to reduce the workflow from days to hours. Our ^M/WExM protocol maintains the superior resolution of the original ExM protocol and yields a higher magnitude of expansion, suggesting that M/W radiation may also facilitate the expansion process. We then adapted the M/W protocol to the whole-mount brain of Drosophila melanogaster fruit flies, and successfully reduced the processing time of a widely used Drosophila IHC-ExM protocol from 6 to 2 days. This demonstrates that with appropriate adjustment of M/W parameters, this protocol can be readily adapted to different organisms and tissue types to greatly increase the efficiency of ExM experiments.