STAR Protocols (Dec 2023)

Optimized protocol for whole-mount RNA fluorescent in situ hybridization using oxidation-mediated autofluorescence reduction on mouse embryos

  • Angela Morabito,
  • Jonas Malkmus,
  • Anna Pancho,
  • Aimée Zuniga,
  • Rolf Zeller,
  • Rushikesh Sheth

Journal volume & issue
Vol. 4, no. 4
p. 102603

Abstract

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Summary: Tissue autofluorescence poses significant challenges for RNA and protein analysis using fluorescence-based techniques. Here, we present a protocol that combines oxidation-mediated autofluorescence reduction with detergent-based tissue permeabilization for whole-mount RNA-fluorescence in situ hybridization (FISH) on mouse embryonic limb buds. We describe the steps for embryo collection, fixation, photochemical bleaching, permeabilization, and RNA-FISH, followed by optical clearing of RNA-FISH and immunofluorescence samples for imaging. The protocol alleviates the need for digital image post-processing to remove autofluorescence and is applicable to other tissues, organs, and vertebrate embryos. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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