In Situ Hybridization of Electoplaque trfRNA

For in situ hybridization (ISH), frozen T. californica electoplaque was cryosectioned (30–40 μm) and mounted onto Superfrost Plus slides (VWR, Radnor, PA). Fluorescein-labeled anti-sense morpholino oligomer probe matching coding 5′-trfRNA^GLU was synthesized from Gene Tools (Philomath, OR). Control fluorescein-labeled anti-sense cRNA probes matching coding 5′-trfRNA^GLU and scrambled were synthesized from IDT (Coralville, IA). Probes were hybridized to sections as previously described54 (link), with minor modifications in amplification strategy. Following overnight hybridization, slides were incubated with alkaline phosphatase conjugated anti-fluorescein antibody (1:5000; 11426338910; Roche Life Sciences, Indianapolis, IN) for overnight at 4 °C. Tissues were washed and incubated in Fast Red (11496549001; Roche Life Sciences) according to manufacturer’s instructions for overnight at 4 °C in dark. Confocal images were captured with a Zeiss 780 Structure Illumination microscope (Zeiss, Germany). Sequences used for probe generation are listed below.
trfRNA^Glu Morpholino: 5′- GCCGAATCCTAACCACTAGACCACC-fluoroscein
trfRNA^Glu DNA control: 5′- GCCGAATCCTAACCACTAGACCACC-fluoroscein
trfRNA^Glu DNA scramble: 5′ - CCCGAATCGTAACGACTAGAGCAGC-fluoroscein