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

agarose stock

 * dissolve agarose powder in E3 in a glass bottle to a final concentration of 0.1/1.5%
 * heat up the mixture in a microwave w/o boiling it
 * distribute 1 ml aliquots in 1.5 ml reaction tubes
 * keep the aliquots in the fridge

before the mounting

 * melt aliquots of agarose in a heat-block at 70 deg Celsius
 * vortex the aliquots
 * keep them in a heat-block at 38 deg Celsius

prepare agarose plates

 * melt aliquots of agarose in a heat-block at 70 deg Celsius
 * vortex the aliquots
 * pour 2 aliquots of agarose into a 35 mm plastic dish

clean FEP tubes

 * 1) wear gloves!
 * 2) (ddH2O got autoclaved and devolatilize)
 * 3) (70% EtOH got devolatilize)
 * 4) flush tubes with 1 M NaOH, use syringe with attached needle
 * 5) transfer flushed tubes to fresh Falcon with 0.5 M NaOH, use forceps
 * 6) put the Falcon in an ultrasonic bath for 10 min
 * 7) from now on, touch the tubes only with gloves and/or forceps!
 * 8) transfer the tubes from the Falcon into small basin with ddH2O and flush them with ddH2O
 * 9) flush the tubes with 70% EtOH
 * 10) transfer the tubes to a fresh Falcon with 70% EtOH
 * 11) put the Falcon in an ultrasonic bath for 10 min
 * 12) transfer the tubes to a fresh Falcon with ddH2O for storage

Short-term imaging I: the classic approach
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.

procedure
If required for multi-view registration, add fluorescent beads to the melted aliquots of agarose. Keep the agarose aliquots 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.

prepare syringes w/ FEP tubes

 * + clean tubes
 * + coat tubes w/ methylcellulose