Imaging the Intestinal Transcriptome With Multiplexed Error‐Robust Fluorescence In Situ Hybridization (MERFISH)

Abstract Multiplexed error‐robust fluorescence in situ hybridization (MERFISH) is a massively multiplexed single RNA–molecule imaging technique capable of spatially resolved single‐cell transcriptomic profiling of thousands of genes in millions of cells within intact tissue slices. Initially introduced for brain tissues, MERFISH has since been extended to other tissues, where rapid RNA degradation during the preparation process can pose challenges. This protocol outlines the application of MERFISH in one such challenging tissue, the mammalian gastrointestinal tract. We describe two complementary protocols leveraging either fresh frozen or fixed frozen approaches and describe methods for combining RNA imaging with immunofluorescence. While these protocols were designed and validated in gut tissues, we anticipate that they will be useful resources for the application to other challenging tissue types. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : Fixed‐frozen sample preparation Basic Protocol 2 : Fresh‐frozen sample preparation Basic Protocol 3 : Encoding probe construction Basic Protocol 4 : MERFISH imaging Basic Protocol 5 : Image decoding Support Protocol 1 : Coverslip silanization Support Protocol 2 : Poly‐ d ‐lysine (PDL) coating of the coverslips Support Protocol 3 : Hybridization buffer preparation Support Protocol 4 : Trolox quinone stock preparation Support Protocol 5 : TCEP stock preparation Alternate Protocol 1 : MERFISH‐compatible immunofluorescent boundary stains in fresh frozen tissue Alternate Protocol 2 : Immunofluorescent boundary stains with methacrylate‐NHS‐anchored antibodies for PFA‐fixed samples Alternate Protocol 3 : Guanidine‐HCl tissue clearing