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 Zúñiga, Rolf Zeller, Rushikesh Sheth
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.