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Abstract BackgroundIn the past few years, there has been an explosion in single-cell transcriptomics datasets, yet in vivo confirmation of these datasets is hampered in plants due to lack of robust validation methods. Likewise, modeling of plant development is hampered by paucity of spatial gene expression data. RNA fluorescence in situ hybridization (FISH) enables investigation of gene expression in the context of tissue type. Despite development of FISH methods for plants, easy and reliable whole mount FISH protocols have not yet been reported. ResultsWe adapt a 3-day whole mount RNA-FISH method for plant species based on a combination of prior protocols that employs hybridization chain reaction (HCR), which amplifies the probe signal in an antibody-free manner. Our whole mount HCR RNA-FISH method shows expected spatial signals with low background for gene transcripts with known spatial expression patterns in Arabidopsis inflorescences and monocot roots. It allows simultaneous detection of three transcripts in 3D. We also show that HCR RNA-FISH can be combined with endogenous fluorescent protein detection and with our improved immunohistochemistry (IHC) protocol. ConclusionsThe whole mount HCR RNA-FISH and IHC methods allow easy investigation of 3D spatial gene expression patterns in entire plant tissues.