Protocol for open-source automated universal high-content multiplex fluorescence for RNA in situ analysis
Jean Descarpentrie1, Florian Bernard2, Wilfried Souleyreau3
1Department of Molecular Biology, 90187 Umeå, Sweden; Umeå Centre for Microbial Research (UCMR), Umeå University, 90187 Umeå, Sweden.
STAR Protocols
|December 7, 2024
Abstract:
In situ hybridization visualizes RNA in cells, but image analysis is complex. We present a protocol based on open-source software for automated high-content multiplex fluorescence in situ transcriptomics analysis. Steps include nuclei segmentation with a Fiji macro and quantification of up to 14 mRNA probes per image. We describe procedures for storing raw data, quality control images, and the use of a Python app to summarize all the results in one spreadsheet detailing the number of single or co-positive cells.
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