Expression of molecular markers in subpopulations of mouse stellate ganglion neurons using single molecule FISH (RNAScope)
Study Purpose: Sympathetic regulation of peripheral organ function is achieved through the coordinated actions of secretomotor, vasomotor and erector sympathetic neurons, in a manner that is highly conserved across species. Functionally distinct classes of sympathetic neurons have been defined by their firing properties and the expression of different combinations of neurotransmitters and neuropeptides. The identification of unique molecular markers for these populations would open the door to the application of genetic tools to parse the actions of secretomotor, vasomotor and erector sympathetic neurons. Here we confirmed the specificity of the expression patterns of the marker genes identified in bioinformatic analyses of single cell RNA-Seq of mouse stellate ganglia (SG) by multiplex single molecule fluorescent in situ hybridization (smFISH) on SG cryosections.
Data Collection: Data consists of 41 images (CZI format) of combinations of RNAScope probes marking distinct populations of SG neurons in C57BL6/J mice using a Zeiss LSM 710 inverted confocal microscope.
Primary Conclusion: Expression of candidate markers for subpopulations of stellate ganglion neurons detected by smFISH closely matched the expression pattern predicted by the scRNA-Seq analyses.
Experimental Design: Mouse ganglia were dissected, fixed in 4% PFA in 0.1M PB overnight at 4°C, washed with Phosphate Buffer Saline (PBS) at 4°C for at least 1 h and encased in Optimal Cutting Temperature compound (Tissue-Tek) then cut into 10 μm cryo-sections on slides, and stored at -80°C. RNAscope single-molecule fluorescent in situ hybridization (smFISH) multiplex version was performed as instructed by Advanced Cell Diagnostics (ACDBio, Document Number 320293-USM) without modification. Expression patterns were visualized by confocal microscopy.
Completeness: This dataset is a part of a larger study, " Expression of molecular markers for SG neuronal subpopulations in 3 sympathetic ganglia."
Subjects & Samples: Male (n=3) and female (n=1) adult mice (RRID:IMSR_JAX:000664) were used in this study.
Primary vs derivative data: Primary data is organized by the subject ID folders and sample ID subfolder. Each sample subfolder contains confocal images (CZI format). Image data in the derivative folder (JPEG2000 and OME-TIFF) was derived from primary images (.CZI).
Important Notes: A complete list of molecular probes is shown in the associated protocol https://www.protocols.io/view/expression-of-molecular-markers-in-subpopulations-b62mrgc6/materials, and is available as a pdf file in the protocol folder in this dataset.
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