Fluorescence hybridization chain reaction enables localization of multiple molecular classes combined with plant cell ultrastructure

Abstract

ABSTRACTBackgroundRecent developments in hybridization chain reaction (HCR) have enabled robust simultaneous localization of multiple mRNA transcripts using fluorescencein situhybridization (FISH). Once multiple split initiator oligonucleotide probes bind their target mRNA, HCR uses DNA base-pairing of fluorophore-labeled hairpin sets to self-assemble into large polymers, amplifying the fluorescence signal and reducing non-specific background. Few studies have applied HCR in plants, despite its demonstrated utility in whole mount animal tissues and cell culture. Our aim was to optimize this technique for sectioned plant tissues embedded with paraffin and methacrylate resins, and to test its utility in combination with immunolocalization and subsequent correlation with cell ultrastructure using scanning electron microscopy.ResultsApplication of HCR to 10 µm paraffin sections of 17-day-oldSetaria viridis(green millet) inflorescences using confocal microscopy revealed that the transcripts of the transcription factorKNOTTED 1(KN1) were localized to developing floret meristem and vascular tissue whileSHATTERING 1(SH1) andMYB26transcripts were co-localized to the breakpoint below the floral structures (the abscission zone). We also used methacrylate de-embedment with 1.5 µm and 0.5 µm sections of 3-day-oldArabidopsis thalianaseedlings to show tissue specificCHLOROPHYLL BINDING FACTOR a/b(CAB1) mRNA highly expressed in photosynthetic tissues andELONGATION FACTOR 1 ALPHA(EF1α) highly expressed in meristematic tissues of the shoot apex. The housekeeping geneACTIN7(ACT7) mRNA was more uniformly distributed with reduced signals using lattice structured-illumination microscopy. HCR using 1.5 µm methacrylate sections was followed by backscattered imaging and scanning electron microscopy thus demonstrating the feasibility of correlating fluorescent localization with ultrastructure.ConclusionHCR was successfully adapted for use with both paraffin and methacrylate de-embedment on diverse plant tissues in two model organisms, allowing for concurrent cellular and subcellular localization of multiple mRNAs, antibodies and other affinity probe classes. The mild hybridization conditions used in HCR made it highly amenable to observe immunofluorescence in the same section. De-embedded semi-thin methacrylate sections with HCR were compatible with correlative electron microscopy approaches. Our protocol provides numerous practical tips for successful HCR and affinity probe labeling in electron microscopy-compatible, sectioned plant material.