During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to successfully complete the first meiotic cell division. Recombinases RAD51 and DMC1 play an important role in homology search, but their mechanistic contribution to this process is not fully understood. Super-resolution, single-molecule imaging of RAD51 and DMC1 provides detailed information on recombinase accumulation on DSBs during meiotic prophase. Here, we present a detailed protocol of recombination foci analysis of three-color direct stochastic optical reconstruction microscopy (dSTORM) imaging of SYCP3, RAD51, and DMC1, fluorescently labeled by antibody staining in mouse spermatocytes. This protocol consists of sample preparation, data acquisition, pre-processing, and data analysis. The sample preparation procedure includes an updated version of the nuclear spreading of mouse testicular cells, followed by immunocytochemistry and the preparation steps for dSTORM imaging. Data acquisition consists of three-color dSTORM imaging, which is extensively described. The pre-processing that converts fluorescent signals to localization data also includes channel alignment and image reconstruction, after which regions of interest (ROIs) are identified based on RAD51 and/or DMC1 localization patterns. The data analysis steps then require processing of the fluorescent signal localization within these ROIs into discrete nanofoci, which can be further analyzed. This multistep approach enables the systematic investigation of spatial distributions of proteins associated with individual DSB sites and can be easily adapted for analyses of other foci-forming proteins. All computational scripts and software are freely accessible, making them available to a broad audience. Key features Preparation of spread nuclei, resulting in a flattened preparation with easy antibody-accessible chromatin-associated proteins on dSTORM-compatible coverslips. dSTORM analysis of immunofluorescent repair foci in meiotic prophase nuclei. Detailed descriptions of data acquisition, (pre-)processing, and nanofoci feature analysis applicable to all proteins that assemble in immunodetection as discrete foci. Graphical overview.
Keywords: DMC1; Meiosis; Nuclear spreading; RAD51; SYCP3; Single-molecule analysis; Spermatocytes; Super-resolution microscopy; dSTORM.
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