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

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.