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Updated: Jul 15, 2025
Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos
Published on: January 30, 2013
Optimized protocol for whole-mount RNA fluorescent in situ hybridization using oxidation-mediated autofluorescence
Angela Morabito1, Jonas Malkmus1, Anna Pancho1
1Developmental Genetics, Department of Biomedicine, University of Basel, 4058 Basel, Switzerland.
Abstract:
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.
