Monitoring Recovery of Laser-Ablated Interneuromast Cells in Zebrafish Using Confocal Microscopy

Abstract

Source: Volpe, B. A., et al. Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells. J. Vis. Exp. (2020)

This video demonstrates monitoring interneuromast cell recovery in zebrafish using confocal microscopy, tracking cell death and repair through time-lapse fluorescence imaging.

Protocol

1. Post-ablation imaging and time-lapse microscopy to study regeneration

Results

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12-well PTFE Printed Slides	Electron Microscopy Sciences	63425-05
15 mM Tricaine stock solution	Sigma	E10521	15 mM Tricaine in reverse osmosis water
1X E3 + 600 µM Tricaine			Dilute 15 mM Tricaine stock 25X in 1X E3 media
1X E3 media			Dilute 60X E3 media to 1X in reverse osmosis water (16.7 ml/L)
60 X E3 media	All components purchased from Sigma-Aldrich		34.4 g Nacl, 1.52 g Kcl, 5.8 g CaCl2.2H2O, 9.8 g MgSO4.7H20 in 1 liter reverse-osmosis water
Bx60 Compound microscope with mercury arc lamp fluorescence	Olympus
LSM 700 confocal microscope equipped with 405 nm, 488 nm, and 555 or 561 nm lasers	Carl Zeiss Microscopy, LLC		A 5 mW 405 nm laser was used for ablation; Ablations and imaging were performed through a 63x Plan-Apochromat objective with an NA of 1.40
FIJI/ImageJ Image processing software	Multiple contributors		Downloadable at https://fiji.sc/
Dissecting needle modified into hair knife	Fisher Scientific	19010	An eyelash was glued onto the end of a wood-handled dissecting needle
Microsoft Excel software	Microsoft Corp.		Google sheets is a no-cost alternative to Microsoft Excel
Glass bottom 35 mm dishes with no. 1.5 coverslip, 20 mm window	Mattek Corporation	P35G-1.5-20-C	35 mm petri dish, 20 mm glass window
Immersol 518F Immersion Oil	Fisher Scientific	12-624-66A
Low Gelling Agarose	Sigma Life Science	A9414-256
Corning Netwell Insert with 74 um Polyester Mesh, 24 mm Insert	Millipore Sigma	CLS3479-48EA
Rstudio software	Rstudio PBC		Downloadable at https://rstudio.com/
SMX-168-BL Stereo microscope	Motic
Transfer Pipette	Fisher Scientific	13-711-7M
ZEN software	Carl Zeiss Microscopy, LLC