Rnascope multiplex fluorescent assay kit

An RNAscope multiplex fluorescent assay kit was used for in situ hybridization (Advanced Cell Diagnostics, Newark, CA). Briefly, formalin-fixed sections were dehydrated in ethanol followed by protease exposure. Sections were then hybridized with RNAscope oligonucleotide probes against Drd2. Following probe hybridization, slides were incubated with signal amplifier according to RNAscope protocols. Slides were then washed with RNAscope wash buffer. Finally, slides were mounted with DAPI counterstain.

To determine the distribution of C1 (PNMT + TH) and A1 (TH only) neurons in mouse medulla oblongata, three mice were anesthetized and perfused transcardially with 3% paraformaldehyde. Brains were removed and postfixed in the same fixative for 16–18 hours at 4 °C. Brains were sectioned and placed in cryoprotectant as described above. Sections were briefly washed in sterile phosphate buffered saline, mounted on charged slides and dried overnight. After 2 rinses in sterile water, sections were incubated with “pretreat 4” from the RNAscope Multiplex Fluorescent Assay kit (Advanced Cell Diagnostics, Hayward, CA) for 30 minutes at 42 °C. Sections were rinsed twice in sterile water and incubated in RNAscope catalog oligonucleotide probes for PNMT, TH for 2 hours at 42 °C. PNMT probes were based on the sequence for mouse PNMT (NM_008890.1) spanning base pairs 2–849. TH probes were based on the sequence for mouse TH (NM_009377.1) spanning base pairs 483–2603. PNMT was used in RNAscope channel 1 and TH in channel 2. After incubation in probes, tissue was treated exactly according to the manufacturer’s protocol (ACD) using Amp 4 Alt A-FL in the final incubation resulting in PNMT transcripts being tagged with Alexa 488 and TH transcripts with Atto 550. Slides were covered with Prolong Diamond Antifade mountant (Molecular Probes, Eugene, OR).

Higher visual area borders of mouse brain were mapped using ISI, and 10nl of scAAVretro-eGFP and 10nl of scAAVretro-mCherry were injected into AL and PM respectively. At 2-3 weeks after virus injections, the animal was sacrificed in a CO₂ chamber and brains were dissected out from the skull, and immediately frozen in Tissue-Tek O.C.T Compound on dry ice. Brains were cryo-sectioned coronally with 20μm thickness using Leica CM 1950 and stored at −80°C until use. smFISH was performed using RNAscope Fluorescent Multiplex Assay kit (Advanced Cell Diagnostics). Sections containing eGFP, mCherry, and smFISH in V1 were imaged with 20X or 63X Zeiss Airyscan 880. Imaging was performed with Z-stack with 1μm optical section and tiling. The number of smFISH puncta on eGFP+ or mCherry+ nuclei was manually counted using ImageJ FIJI. Wilcoxon rank-sum test was used for statistical analysis.