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Zebrafish embryo preparation for imaging in OpenSiMView light-sheet microscope .md

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Zebrafish embryo preparation for imaging in OpenSiMView light-sheet microscope

This protocol describes the preparation of zebrafish embryos for imaging in a OpenSimView light-sheet microscope. The embryos are dechorionated, transferred into FEP tubes embedded in low gelling temperature agarose, and mounted in a custom-made sample capillary holder that is placed in a mounting chamber. Filtered embryo water with tricaine is circulated in the chamber for imaging after 15 hpf.

Materials:

  • Embryos
  • Sharp forceps
  • Binocular dissecting microscope
  • FEP tubes (Zeus Inc. custom order)
  • 0.1% low gelling temperature agarose (Sigma, A0701)
  • Custom-made sample capillary holder
  • 0.7% low-gelling temperature agarose
  • Holder insertion tool
  • Section pipette tip
  • Low toxicity silicone (Kwik-Siltm from World Precision Instrument)
  • Filtered embryo water
  • 0.016% tricaine

Protocol:

  1. Gently dechorionate the embryos using a pair of sharp forceps under a binocular dissecting microscope.
  2. Transfer the embryos into FEP tubes embedded in 0.1% low gelling temperature agarose.
  3. Place the FEP tubes in a custom-made sample capillary holder that has been previously filled with 0.7% low-gelling temperature agarose to support the embryo.
  4. Use a holder insertion tool and section pipette tip sealed with low toxicity silicone (Kwik-Siltm from World Precision Instrument) to insert the FEP tube with the embryo into the mounting chamber.
  5. Follow the detailed procedure for mounting, as shown in Supp. Fig. 6a.
  6. Use filtered embryo water with 0.016% tricaine to circulate in the chamber for recording after 15 hpf.
  7. Use CAD drawings of the custom-made FEP holder and the mounting chamber, available in Supp. Fig. 5, for reference.