title	layout	description
Zebrafish Embryo Sample Preparation	page	Zebrafish embryo sample preparation

Zebrafish embryos should be mounted according to the imaging needs, e.g.:

for short-term imaging: in 1.5% agarose inside a glass capillary, pushed out for imaging (the classic approach), w/ optional FEP tube support
for long-term imaging: in 0.1% agarose inside a polymer (FEP) tube
for long-term cardiac imaging: in 3% methylcellulose inside a polymer (FEP) tube

See the links to publications dealing with sample mounting for light sheet microscopy at the bottom of this page.

Material

List


	product	external link	short-term imaging I	short-term imaging II	long-term imaging	long-term cardiac imaging
E3 fish medium	-	Zebrafish: a practical approach (Nüsslein-Volhard and Dahm, 2002)	o	o	o	o
Tricaine/MS-222/TS 222	Aldrich/Sigma E10521	Sigma-Aldrich	o	o	o	o
agarose, low gelling temperature	BioReagent/Sigma A9414	Sigma-Aldrich	o	o	o	o
methyl cellulose	Sigma M0387	Sigma-Aldrich	-	-	-	o
glass capillaries	1) Brand 701904 glass capillaries 20 µl black (accessory/spare part for Transferpettor) or 2) Wheaton 851322 glass capillaries 25 µl, 1 mm diameter, 125 mm in length	1)Brand 2)Wheaton	o	-	-	-
teflon-coated plungers	1) Brand 701932 teflon-coated plungers 10µl/Dig.25µl (accessory/spare part for Transferpettor) or 2) Wheaton 851331 teflon-coated plungers	1) Brand 2)Wheaton	o	-	-	-
1 ml syringe	1) B. Braun Omnifix F Solo 1 ml Syringe or 2) BD tuberculin syringe 309659, 1ml	1) B. Braun 2)BD	-	o	o	o
hypodermic needle	B. Braun 100 Sterican, blunt	B. Braun	-	o	o	o
FEP tubes	Bola S1815-04, inner diameter 0.8 mm, outer diameter 1.6 mm	Bola	-	o	o	o

Prepare 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

Prepare agarose for 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

{% include image src="Heat-block_70.JPG" width="100%" caption="Melt aliquots of agarose at 70 deg Celsius" %}

{% include image src="Vortex.JPG" width="100%" caption="Vortex the aliquots" %}

{% include image src="Heat-block_38.JPG" width="100%" caption="Keep the agarose 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

{% include image src="Heat-block_70.JPG" width="100%" caption="Melt aliquots agarose at 70 deg Celsius" %}

{% include image src="Vortex.JPG" width="100%" caption="Vortex the aliquots" %}

{% include image src="Small_agarose_plate.png" width="100%" caption="Pour 2 agarose aliquots into a 35 mm plastic dish" %}

Prepare methylcellulose stock

Prepare methylcellulose for mounting

Clean & cut FEP tubes

flush tubes with 1 M NaOH, use syringe with attached needle
transfer flushed tubes to fresh Falcon with 0.5 M NaOH, use forceps
put the Falcon in an ultrasonic bath for 10 min
from now on, touch the tubes only with gloves and/or forceps!
transfer the tubes from the Falcon into small basin with ddH2O and flush them with ddH2O
flush the tubes with 70% EtOH
transfer the tubes to a fresh Falcon with 70% EtOH
put the Falcon in an ultrasonic bath for 10 min
cut the tube to small pieces of appropriate length
transfer the tubes to a fresh Falcon with ddH2O for storage

{% include image src="Small_fep_flush.png" width="100%" caption="Flush the FEP tube w/ NaOH/EtOH/ddH2O" %}

{% include image src="Small_fep_cut.png" width="100%" caption="Cut the tube to small pieces of appropriate length" %}

Prepare syringes w/ FEP tubes

take a cleaned and cut FEP tube and carefully attach it to the needle
do not touch the part of the tube that will later be used for imaging

{% include image src="Syringe_unpacked.JPG" width="100%" caption="Braun Omnifix F Solo 1 ml Syringe w/ attached needle" %}

{% include image src="Fep_pipette.JPG" width="100%" caption="Take a cleaned & cut FEP tube..." %}

{% include image src="Fep_needle01.JPG" width="100%" caption="... and attach it..." %}

{% include image src="Fep_needle02.JPG" width="100%" caption="... to the needle" %}

{% include image src="Syringe_fep_attached.JPG" width="100%" caption="Syringe w/ needle and FEP tube" %}

Coat tubes w/ methyl cellulose

slowly take up 3% methyl cellulose with a needle-attached FEP tube
slowly push out the methyl cellulose and discard it
immediately proceed with mounting

Prepare Zebrafish embryos

{% include image src="Dechorionation_macro.JPG" width="100%" caption="Dechorionation I" %}

{% include image src="Dechorionation_micro02.JPG" width="100%" caption="Dechorionation II" %}

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.

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.

{% include image src="Sample_uptake01.JPG" width="100%" caption="Transfer a Zebrafish embryo of choice from the dish..." %}

{% include image src="Sample_uptake02.JPG" width="100%" caption="... to the melted Agarose" %}

Short-term imaging II: FEP tube as additional support

take up an embryo w/ a FEP tube which is attached to a needle and syringe
wait a couple of minutes for the agarose to solidify and cut off the tube from the needle
store the prepared sample in a 1.5 ml reaction tube filled with E3 fish medium
quickly proceed w/ imaging

{% include image src="Sample_uptake01.JPG" width="100%" caption="Transfer a Zebrafish embryo of choice from the dish..." %}

{% include image src="Sample_uptake02.JPG" width="100%" caption="... to the melted Agarose" %}

{% include image src="Sample_uptake03.JPG" width="100%" caption="Take up the embryo into the FEP tube" %}

{% include image src="Fep_with-fish_macroA.JPG" width="100%" caption="FEP w/ Zebrafish embryo" %}

{% include image src="Fep_with-fish_macroB.JPG" width="100%" caption="Check the orientation of the embryo in the tub" %}

{% include image src="Fep_with-fish_micro.JPG" width="100%" caption="Zebrafish embryo in FEP tube" %}

{% include image src="Fep-prep.JPG" width="100%" caption="FEP tubes w/ Zebrafish embryos in 1.5% agarose" %}

{% include image src="Fep_cut.JPG" width="100%" caption="Cut the tube off the needle" %}

{% include image src="Fep_with-fish_cutted_micro.JPG" width="100%" caption="Check the orientation of the embryo under a stereo microscope" %}

{% include image src="Fep_zeiss-holder.JPG" width="100%" caption="The prepared sample in a holder for light sheet microscopy" %}

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.

{% include image src="Sample_uptake01.JPG" width="100%" caption="Transfer a Zebrafish embryo of choice from the dish..." %}

{% include image src="Sample_uptake02.JPG" width="100%" caption="... to the melted Agarose" %}

{% include image src="Sample_uptake03.JPG" width="100%" caption="Take up the embryo into the FEP tube" %}

{% include image src="Fep_with-fish_macroA.JPG" width="100%" caption="FEP with Zebrafish embryo" %}

{% include image src="Fep_with-fish_macroB.JPG" width="100%" caption="Check the orientation of the embryo in the tube" %}

{% include image src="Fep_with-fish_micro.JPG" width="100%" caption="Zebrafish embryo in FEP tube" %}

{% include image src="Fep_cut.JPG" width="100%" caption="Cut the tube off the needle" %}

{% include image src="Fep_plugA.JPG" width="100%" caption="Carefully stick the tube into the prepared agarose plates" %}

{% include image src="Fep_plugB.JPG" width="100%" caption="Take out the tube w/ a little rotational move to keep a plug inside" %}

{% include image src="Fep_with-fish_cutted_micro.JPG" width="100%" caption="Check the orientation of the embryo under a stereo microscope" %}

{% include image src="Fep_zeiss-holder.JPG" width="100%" caption="The prepared sample in a holder for light sheet microscopy" %}

Long-term cardiac imaging