Zebrafish embryo sample preparation

Zebrafish embryos should be mounted according to the imaging needs, e.g.: Follow the external links for publications dealing with sample mounting for light sheet microscopy.
 * 1) for short-term imaging: in 1.5% agarose inside a glass capillary, pushed out for imaging (the classic approach), w/ optional FEP tube support
 * 2) for long-term imaging: in 0.1% agarose inside a polymer (FEP) tube
 * 3) for long-term cardiac imaging: in 3% methylcellulose inside a polymer (FEP) tube

Short-term imaging
The method of choice mounting Zebrafish embryos is very similar to the one for mounting Drosophila embryos. It is simple and straight-forward, but restricts the embryo from growing. This limits the usable time frame for imaging to about 1-2 hours from the mounting.

material

 * glass capillaries 20 µl black (Brand 701904) (accessory/spare part for Transferpettor)
 * Teflon-coated plungers 10µl/Dig.25µl (Brand 701932)
 * low gelling temperature agarose (BioReagent/Sigma A9414)
 * E3 (Nüsslein-Volhard and Dahm, 2002)
 * Tricaine (Aldrich)

procedure
Prepare low-melting point agarose in fish medium (E3, without Methylene Blue) at a final concentration of 1.5%. Add fluorescent beads if required. Melt it at 70 deg Celsius and keep it at 38 deg Celsius. Transfer a Zebrafish embryo of choice to the agarose and take it up with a glass capillary (inner diameter around 1 mm) and a plunger. Let the agarose solidify and keep the embryo in the capillary submerged in fish medium. For imaging, push out the part of the agarose which contains the embryo. If required, add Tricaine to the imaging chamber.

Long-term imaging
The limitations of mounting a zebrafish embryo in 1.5% agarose can be overcome by using a lower concentration of agarose plus a transparent solid support. To achieve unpersuaded image quality, the refractive index of the support has to match that of the medium. A good choice is FEP (fluorinated ethylene propylene). This polymer has a refractive index close to the one of water, and can be ordered as tube in various diameters right from the shelf. For imaging, the Zebrafish embryo is kept inside the cleaned and coated tube, embedded in 0.1% agarose with some Tricaine.

material

 * FEP tubes (Bola S1815-04, inner diameter 0.8 mm, outer diameter 1.6 mm)
 * B. Braun Omnifix F Solo 1 ml Syringe
 * B. Braun needle (100 Sterican, blunt)
 * low gelling temperature agarose (BioReagent/Sigma A9414)
 * methylcellulose (Sigma)
 * E3 (Nüsslein-Volhard and Dahm, 2002)
 * Tricaine (Aldrich)

prepare agarose

 * + prepare agarose plates
 * + prepare methylcellulose

prepare syringes w/ FEP tubes

 * + clean tubes
 * + coat tubes w/ methylcellulose

material

 * FEP tubes (Bola S1815-04, inner diameter 0.8 mm, outer diameter 1.6 mm)
 * B. Braun Omnifix F Solo 1 ml Syringe
 * B. Braun needle (100 Sterican, blunt)
 * methylcellulose (Sigma)
 * low gelling temperature agarose (BioReagent/Sigma A9414)
 * E3 (Nüsslein-Volhard and Dahm, 2002)
 * Tricaine (Aldrich)