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Intubation-based anesthesia for long-term time-lapse imaging of adult zebrafish

Nature Protocols volume 10, pages 2064–2073 (2015)Cite this article

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Abstract

Zebrafish possess the remarkable ability to regenerate a vast variety of tissues, even as adults. However, direct imaging of regenerative processes in adult zebrafish remains challenging because of the lack of suitable anesthesia protocols. Here we present a description of an intubation-based anesthesia procedure that we developed to enable us to image regenerating zebrafish fins and which can be used to continuously anesthetize adult zebrafish for up to 2 d. Fish are immobilized in an imaging chamber followed by oral intubation. Subsequent delivery of anesthetic-containing water is achieved via a peristaltic pump. The setup of the system will take ∼90 min for two adult zebrafish, and it requires only a little previous experience of working with zebrafish. Our protocol will enable the imaging of regenerative processes in the fin and other tissues, and the investigation of processes that require long-term anesthesia, such as immune responses and surgical procedures.

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Figure 1: Schematic of the imaging setup for long-term time-lapse imaging of adult zebrafish.
Figure 2: Imaging chamber for live imaging of adult zebrafish.
Figure 3: Detailed description of imaging setup construction.
Figure 4: Description of embedding an adult zebrafish in the imaging chamber.
Figure 5: Time-lapse imaging of regenerating fin reveals cell migration during early fin regeneration.

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Acknowledgements

This work was funded by the Max Planck Society (A.F.S.), the Deutsche Forschungsgemeinschaft (DFG SI-1374/3-1; A.F.S.), the Sonderforschungsbereich (SFB) 629 and a European Research Council (ERC) starting grant (260794-ZebrafishAngio; A.F.S.). This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Cells-in-Motion Cluster of Excellence (EXC 1003-CIM), University of Münster, Germany. We are grateful to G. Weidinger and F. Knopf for sharing their expertise in long-term imaging of fin regeneration. We thank M. Bradter for help with movie processing.

Author information

Authors and Affiliations

  1. Max Planck Institute for Molecular Biomedicine, Muenster, Germany

    Cong Xu, Stefan Volkery & Arndt F Siekmann

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  1. Cong Xu
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  2. Stefan Volkery
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Contributions

C.X. designed the protocol. C.X. and S.V. designed the imaging chamber. S.V. and A.F.S. commented on the procedures. C.X., S.V. and A.F.S. wrote the manuscript.

Corresponding author

Correspondence to Arndt F Siekmann.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Detailed description of setup of pressure monitoring system.

To be assembled by an electrician

Housing small distribution board AK03, Part#73540301, manufacturer: Spelsberg AK A2.2 Schuko plug, Part# 10754, manufacturer Mennekes, non-metallic sheathed cable 3 way NYM-J 32x1,5 T2.3 power supply for DIN-rails, 120 - 370 V ac, 90-265 V ac, 24Vdc 0,42A, Part# 1SVR427041R0000 - CP-D 24/0.42, manufacturer: ABB K2.5 monostable relay for DIN-rails, 24Vdc/1,25W coil, two way closer, 20A/250Vac, Part#: 22.22.9.024.4000 manufacturer: Finder K2.6 monostable relay for DIN-rails, 24Vdc coil, two way opener, 20A/250Vac, Part#: 22.24.9.024.4000 manufacturer: Finder P2.4 pressure switch, Part#DS-E1V10F4B2PR00, manufacturer: Norgren, connector plug M12x1, Part# 0523057, manufacturer: Norgren A2.7 Schuko wall socket; Part# 620W, manufacturer: Jung, non-metallic sheathed cable 3 way NYM-J 32x1,5 Explanation of setup: A2.2 provides 230V AC power input. T2.3 supplies P2.4, K2.5 and K2.6 with 24V DC. In the case the monitored pressure is lower than switching point 1 (SP1), K2.5 closes activating output of 230 V AC at A2.7. K2.5 opens again deactivating output of 230 V AC at A2.7 as the monitored pressure is below re-switching point 1 (rP1). This ensures the peristaltic pump plugged into A2.7 is only active when the vacuum pump is active. In case of vacuum pump failure, A2.7 has no output power and the peristaltic pump stops. In case the monitored pressure is lower than SP2, K2.6 opens deactivating output of 230 V AC at A2.7. K2.6 closes again activating output of 230 V AC at A2.7 as the monitored pressure is below rP2. This ensures that the peristaltic pump is switched off in case the soaking tubing to remove surplus of water in the mold is blocked e.g. by a piece of agarose.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 (PDF 220 kb)

Preparation of Tricaine. (MP4 15558 kb)

Live imaging setup. (MP4 28586 kb)

Embedding adult zebrafish.

The movie illustrates a typical in vivo imaging acquisition session. Here test images are recorded to check for adequate image signal and to optimize the microscope settings (Step 45). All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital and are in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. (MP4 28940 kb)

41596_2015_BFnprot2015130_MOESM457_ESM.mp4

Overview of the imaging setup and transfer of imaging chamber into the incubation chamber of the SP5 confocal microscope. (MP4 13463 kb)

Time-lapse imaging of regenerating fin reveals cell migration during early fin regeneration. (AVI 8697 kb)

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Xu, C., Volkery, S. & Siekmann, A. Intubation-based anesthesia for long-term time-lapse imaging of adult zebrafish. Nat Protoc 10, 2064–2073 (2015). https://doi.org/10.1038/nprot.2015.130

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