研究目的
To monitor MET channel- and presynapse-dependent changes in calcium within individual hair cells and among cells within neuromasts in larval zebrafish using a genetically encoded calcium indicator, GCaMP6s.
研究成果
The protocol outlined provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal. It is complementary to studies of mammals and can be used as a platform to test the efficacy of genetically encoded indicators for examining activity in mammalian hair cells.
研究不足
The preparation only remains robust for 1-3 hours after it is established. Photobleaching and phototoxicity can occur after repeated imaging trials. The technique is sensitive to motion artifacts, requiring a reasonable amount of practice and skill for proper execution.
1:Preparation of Solutions:
Includes the preparation of α-bungarotoxin, embryo buffer (E3), neuronal buffer (NB), and MS-222 stock for anesthesia.
2:Preparation of Imaging Chamber and Pins:
Details the setup of the imaging chamber and the creation of pins for immobilizing larvae.
3:Preparation of Needles for Paralysis and Stimulation:
Describes the preparation of heart injection needles and fluid-jet needles.
4:Pinning and Immobilizing Larva to Imaging Chamber:
Outlines the process of immobilizing the larva on the imaging chamber.
5:Injection of α-Bungarotoxin into the Heart Cavity to Paralyze Larva:
Details the method for paralyzing the larva to prevent movement during imaging.
6:Preparation of Microscope and Fluid-jet Setup:
Describes the assembly of the confocal microscope and fluid-jet setup for stimulation.
7:Alignment of Larva and Fluid-jet:
Explains the alignment process to ensure accurate stimulation and imaging.
8:Imaging Acquisition Procedure Option
Single-plane Acquisition: Details the settings and procedures for single-plane imaging.
9:Imaging Acquisition Procedure Option
Multi-plane Acquisition: Describes the settings and procedures for multi-plane imaging.
10:Control:
Pharmacological Block of All Evoked Calcium Signals: Outlines the use of BAPTA to block calcium signals as a control.
11:Control:
Pharmacological Block of Presynaptic Calcium Signals (Optional): Describes the optional use of isradipine to block presynaptic calcium signals.
12:Image Processing and Graphical Representation of Data:
Explains the use of Fiji for image processing and data representation.
13:Image Processing and Heat Map Representation of Spatio-temporal Calcium Signals:
Details the creation of heat maps for visualizing calcium signals.
14:Image Processing and Spatio-temporal Heat Map Representation Using a Fiji Macro:
Describes the use of a Fiji macro for automated heat map creation.
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获取完整内容-
GCaMP6s
Genetically encoded calcium indicator used to measure calcium signals in zebrafish lateral-line hair cells.
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confocal microscope
Used for imaging calcium signals in vivo.
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fluid-jet
Used to stimulate lateral-line hair cells in a controlled and reproducible manner.
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α-bungarotoxin
Used to paralyze zebrafish larvae during functional imaging.
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MS-222
Anesthetic used to immobilize larvae during pinning and α-bungarotoxin injection.
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BAPTA
Used to cleave the tip links required to gate apical MET channels located in hair bundles as a control.
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isradipine
Used to block the L-type calcium channels at the hair-cell presynapse as an optional control.
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Fiji
Open source analysis software used for image processing and data representation.
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