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

Recent developments in hybridization chain reaction (HCR) have enabled robust simultaneous localization of multiple mRNA transcripts using fluorescence in situ hybridization (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. Results: Application of HCR to 10 μm paraffin sections of 17-day-old Setaria viridis (green millet) inflorescences using confocal microscopy revealed that the transcripts of the transcription factor KNOTTED 1 (KN1) were localized to developing floret meristem and vascular tissue while SHATTERING 1 (SH1) and MYB26 transcripts 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-old Arabidopsis thaliana seedlings to show tissue specific CHLOROPHYLL BINDING FACTOR a/b (CAB1) mRNA highly expressed in photosynthetic tissues and ELONGATION FACTOR 1 ALPHA (EF1α) highly expressed in meristematic tissues of the shoot apex. The housekeeping gene ACTIN7 (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. Conclusion: HCR 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. ### Competing Interest Statement The authors have declared no competing interest.