研究目的
To develop a high-throughput, multiplexable fluorescence in situ hybridization (FISH) platform for identifying neurotransmitter cell types in the intact Drosophila CNS, enabling reliable detection of eight different neurotransmitter phenotypes.
研究成果
The FISH platform successfully detects eight neurotransmitter phenotypes in Drosophila brains, with high throughput and multiplexing capabilities. It provides reliable mapping to specific cell types and can be applied to developmental studies and other gene expression analyses, offering a valuable tool for neuroscience research.
研究不足
Expression of markers does not always indicate transmitter phenotype (e.g., neurons may acquire transmitters via uptake); not all potential transmitters are covered (e.g., glycine); limitations in signal strength for precise counts in some neurons.
1：Experimental Design and Method Selection:
The study uses FISH to detect mRNA markers for neurotransmitters in whole-mount Drosophila brains, with optimizations for high-throughput processing and multiplexing.
2：Sample Selection and Data Sources:
Drosophila fly stocks, including GAL4 driver lines and mutants, were used; samples were dissected from adult and developmental stage flies.
3：List of Experimental Equipment and Materials:
Includes confocal microscopes (Zeiss LSM models), FISH probes labeled with specific fluorophores, HaloTag ligands, coverslips, hybridization chambers, and various chemicals like paraformaldehyde and ethanol.
4：Experimental Procedures and Operational Workflow:
CNS preparation involves dissection, fixation, labeling with HaloTag ligands, dehydration, and mounting on coverslips. FISH protocol includes hybridization in custom chambers, washing, and imaging.
5：Data Analysis Methods:
Confocal imaging with Zeiss ZEN software, image processing with Fiji software, and statistical analysis of cell counts.
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Confocal Microscope
LSM 710
Zeiss
Imaging samples with high resolution for FISH analysis.
ZEISS LSM 990 Spectral Multiplex
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Confocal Microscope
LSM 780
Zeiss
Imaging samples with high resolution for FISH analysis.
ZEISS LSM 990 Spectral Multiplex
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Confocal Microscope
LSM 880
Zeiss
Imaging samples with high resolution for FISH analysis.
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Software
ZEN
Zeiss
Controlling confocal microscopes and processing images.
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Fluorophore
AF594
Thermo Fisher
Labeling FISH probes for detection.
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Fluorophore
DL550
Thermo Fisher
Labeling FISH probes for detection.
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Antibody
mouse anti-serotonin
Thermo Fisher Scientific
Immunostaining for serotonin detection.
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Secondary Antibody
AF568 goat anti-mouse
Thermo Fisher Scientific
Secondary labeling for immunostaining.
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Software
Fiji
Processing and analyzing confocal images.
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HaloTag Ligand
JF646
Labeling HaloTag proteins for imaging.
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HaloTag Ligand
AF488
Promega
Labeling HaloTag proteins for imaging.
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HaloTag Ligand
ATTO 647N
Labeling HaloTag proteins for imaging.
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Fluorophore
Cy3
GE
Labeling FISH probes for detection.
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Fluorophore
Cy5
GE
Labeling FISH probes for detection.
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Fluorophore
Quasar 570
LGC Biosearch Technologies
Labeling FISH probes for detection.
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Fluorophore
CF594
Biotium
Labeling FISH probes for detection.
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Fluorophore
CAL Fluor 610 Red
LGC Biosearch Technologies
Labeling FISH probes for detection.
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Jar
W900180-6
Wheaton
Holding solutions for sample processing.
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Medium
S01416
Sigma Aldrich
Used in dissection and fixation steps.
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