Paolo Cadinu, Evan Yang, Rosalind J. Xu, Brianna R. Watson, Josh Luce, Jeffrey R. Moffitt
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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.</p><p><b>Basic Protocol 1</b>: Fixed-frozen sample preparation</p><p><b>Basic Protocol 2</b>: Fresh-frozen sample preparation</p><p><b>Basic Protocol 3</b>: Encoding probe construction</p><p><b>Basic Protocol 4</b>: MERFISH imaging</p><p><b>Basic Protocol 5</b>: Image decoding</p><p><b>Support Protocol 1</b>: Coverslip silanization</p><p><b>Support Protocol 2</b>: Poly-<span>d</span>-lysine (PDL) coating of the coverslips</p><p><b>Support Protocol 3</b>: Hybridization buffer preparation</p><p><b>Support Protocol 4</b>: Trolox quinone stock preparation</p><p><b>Support Protocol 5</b>: TCEP stock preparation</p><p><b>Alternate Protocol 1</b>: MERFISH-compatible immunofluorescent boundary stains in fresh frozen tissue</p><p><b>Alternate Protocol 2</b>: Immunofluorescent boundary stains with methacrylate-NHS-anchored antibodies for PFA-fixed samples</p><p><b>Alternate Protocol 3</b>: Guanidine-HCl tissue clearing</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 3","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Imaging the Intestinal Transcriptome With Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH)\",\"authors\":\"Paolo Cadinu, Evan Yang, Rosalind J. 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引用次数: 0
Imaging the Intestinal Transcriptome With Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH)
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
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