Expansion microscopy is a powerful technique, which increases the effective resolution of a fluorescence microscope by physically enlarging the specimen within a swellable polymer matrix. When combined with confocal microscopy, it enables affordable and easy-to-implement super-resolution imaging of protein localization in an entire cellular volume. Here, we describe in detail a protocol for the expansion of Euglenozoa, such as Trypanosoma brucei and Euglena gracilis, which achieves an average expansion factor of 4.6 in the case of T. brucei and 3.5 in the case of E. gracilis. This method is compatible with a broad range of available antibodies targeting endogenous proteins or small epitope tags. Additionally, we demonstrate the use of a fluorescent NHS ester for whole-proteome staining, BODIPY lipid stain, and the SYTOX nucleic acid stain for reproducible DNA labeling in expanded cells of these organisms. These reagents further increase the versatility of this simple yet robust super-resolution approach for studies of the cell biology of Euglenozoa.

Department of Parasitology Faculty of Science BIOCEV Charles University Vestec Czech Republic

Laboratory of Cell Motility Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

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