Alkaline phosphatase conjugated anti fluorescein antibody

For in situ hybridization (ISH), mice were euthanized with
CO₂. Lumbar L4–L6 DRGs were dissected and immediately frozen in
OCT on dry ice. Tissue was cryosectioned (10–12 μm), mounted onto
Superfrost Plus slides (VWR, Radnor, PA), frozen at −80°C. Digoxigenin-
and fluorescein-labeled anti-sense cRNA probes matching coding (Gprc5b, Lpar3,
TdTomato, Ntrk2 [Trkb], Prkcq, Nppb, Il31ra) or untranslated regions were
synthesized, hybridized to sections, and visualized as previously described (Liberles and Buck, 2006 (link)), with minor
modifications in amplification strategy. Following overnight hybridization, slides
were incubated with peroxidase conjugated anti-digoxigenin antibody (Roche Applied
Sciences, Indianapolis, IN, USA; 1:200) and alkaline phosphatase conjugated
anti-fluorescein antibody (Roche Applied Sciences, 1:200) for 1 hr at room
temperature. Tissues were washed and incubated in TSA-PLUS-Cy5 (Perkin Elmer)
followed by HNPP (Roche Applied Sciences) according to manufacturer's instructions.
Epifluorescence images were captured with a Leica TCS SP5 II microscope (Leica
microsystems, Buffalo Grove, IL). Sequences of primers used for probe generation are
listed in Table 3.

The process from re-fixation of sections to the chromogenic reaction with NBT/BCIP was performed as described above, except for hybridization. During hybridization, DIG-labeled RNA probes and fluorescein-labeled RNA probes were mixed and mounted simultaneously. After the first chromogenic reaction with NBT/BCIP, the anti-DIG antibody was detached using 100 mM glycine (pH 2.2). After washing in PBS, fluorescein-labeled probes were detected immunohistochemically with an alkaline phosphatase-conjugated anti-fluorescein antibody (1:1,000; Roche, Basel, Switzerland). To visualize the signals, the second color chromogenic reactions were performed at room temperature with SIGMAFAST Fast Red TR/Naphthol AS-MX tablets (Sigma-Aldrich, St. Louis, MO, United States cat#F4523) for the following durations: 5-HTR1A and TPH2, 3 h and 18 h; 5-HTR1B and TPH2, 3–16 h and 18–18.5 h; 5-HTR1D and TPH2, 16 h and 19.3 h; 5-HTR1E and TPH2, 2.5 h and 17.3 h; 5-HTR5A and TPH2, 2.5 h and 17.3 h. Sense probes were used as negative controls for every experiment.

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