RNA probe preparation and in situ hybridization

Forward and reverse universal primers corresponding to regions 533 and 907 nt of bacterial 16S rRNA was used to amplify bacterial 16S rRNA from fed nymphal gut and amplicons cloned into the pGEM Teasy vector (Promega) and verified by sequencing. The Plasmid was linearized to generate sense and antisense RNA by in vitro transcription using T7 or SP6 polymerase with the Hi Scribe RNA synthesis kit (New England Biolabs) and Digoxigenin-11-UTP (Roche) according to the manufacturer’s instructions. Nymphal ticks fed for 48 h were dissected in MEMFA (1M MOPS, 20 mM EGTA, 10 mM MgSO₄, 38% Formaldehyde) fixed for one hour, dehydrated in 100% methanol and used to assess presence of bacterial RNA in guts by whole-mount in situ hybridization using Digoxigenin-labeled sense or antisense essentially as described for Xenopus embryos⁷². Hybridized RNA was detected with alkaline phosphatase-conjugated anti-Digoxigenin antibody (Sigma-Aldrich) and the substrate BM purple (Roche). Guts were visualized using a bright field microscope at 10 X magnification.