RNAscope Fluorescent Multiplex Assay for Spinal Cord

The spinal cord sections were processed according to the manufacturer’s instructions in the RNAscope Fluorescent Multiplex Assay v2 manual for fixed frozen tissue (Advanced Cell Diagnostics), and coverslipped with Fluoromount-G antifade reagent (Southern Biotech) with DAPI (Molecular Probes)^{47 (link),66 (link)}. The following probes, purchased from Advanced Cell Diagnostics were used: Grpr (nucleotide target region 463-1596; GenBank: NM_008177.2), Tac2 (nucleotide target region 15-684; GenBank: NM_009312.2), Vglut2 (nucleotide target region 1986-2998; GenBank: NM_080853.3), Vgat (nucleotide target region 894-2037; GenBank: NM_009508.2), and Npy1r (nucleotide target region: 227-1169; GenBank: NM_010934.4). Sections were subsequently imaged under a Nikon C2+ confocal microscope (Nikon Instruments, Inc.) in three channels with a 20× objective lens. Positive signals were identified as three punctate dots present in the nucleus and/or cytoplasm if the signal was shown up in small dots rather than filling up entire neurons. For co-localization studies, dots associated with single DAPI stained nuclei were assessed as being co-localized. Cell counting was done by a person who was blinded to the experimental design.

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Chen S., Gao X.F., Zhou Y., Liu B.L., Liu X.Y., Zhang Y., Barry D.M., Liu K., Jiao Y., Bardoni R., Yu W, & Chen Z.F. (2020). A spinal neural circuitry for converting touch to itch sensation. Nature Communications, 11, 5074.

Publication 2020

Fluoromount-G antifade reagent DAPI Nikon C2+ confocal microscope

Assay Cell Confocal microscope Cytoplasm Dapi Diagnostics Frozen Lens Molecular probes Neurons Nucleotide Nucleus Single nuclei Spinal cord

Corresponding Organization :

Other organizations : Washington University in St. Louis, Renji Hospital, University of Modena and Reggio Emilia

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Variable analysis

independent variables

RNAscope Fluorescent Multiplex Assay v2 manual for fixed frozen tissue (Advanced Cell Diagnostics)
Probes used: Grpr, Tac2, Vglut2, Vgat, Npy1r

dependent variables

Expression levels of Grpr, Tac2, Vglut2, Vgat, Npy1r

control variables

Sections were coverslipped with Fluoromount-G antifade reagent (Southern Biotech) with DAPI (Molecular Probes)
Positive signals were identified as three punctate dots present in the nucleus and/or cytoplasm
Cell counting was done by a person who was blinded to the experimental design

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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