2 Materials
For all solutions, ultrapure sterile water is used (prepared by purifying deionized water, 18 MΩ-cm at 25
C, and autoclaving). Store
all solutions at room temperature unless otherwise indicated.
2.1 Microscopy
Setup
1. Upright compound microscope with a 20Â or 40Â high
numerical aperture dipping lens (see Note 1).
2. Having a motorized stage will allow imaging several embryos in
every session greatly enhancing experimentation efficiency.
This is especially important, when working with genetic condition in which not all specimens are carriers. Having said that, a
motorized stage is not strictly required.
3. Temperature regulation. This can be obtained by having a
temperature chamber enclosing the microscope head, or by
having a tight regulation of the temperature of the room. In
either case, make sure to stabilize temperature of all the components for at least 1 h before initiating imaging, and that the
temperature is the same at every imaging session.
4. Confocal or spinning disk heads. The use of a spinning disk
head will greatly reduce the time of imaging, allowing to image
a greater number of embryos per session and reducing phototoxicity.
2.2 Mounting
Embryos and Imaging
1. E3 fish medium. Prepare a 60Â stock solution (1 L): 17.2 g of
NaCl, 0.76 g of KCl, 2.9 g of CaCl 2 ·2H 2 O, 4.9 g of MgSO 4 ·7H 2 O. Adjust volume to 1 L of water. Dilute to a 1Â working
solution as needed.
2. Tricaine (MS-222, Sigma A5040-100G). Prepare a 15 mM
stock solution: Add 0.2 g of tricaine powder and 1 mL of
Tris–HCl 1 M pH 9.5 to 49 mL dH 2 O. Adjust pH to 7 and
store 1 mL aliquots at À20
C. After thawing, tricaine aliquots
can be stored at 4
C for a few days (no more than 1 week).
3. 1% Agarose in 1Â E3.
4. 0.6% Low Melting Point (LMP) agarose in 1Â E3 medium.
5. 3% Methyl cellulose (Sigma M-0387, Viscosity 1500 cPs).
Heat 100 mL 1Â E3 in a closed bottle to 60
C, add 3 g methyl
cellulose and mix with a magnetic stirrer. The solution will take
a long time (overnight or longer) to dissolve and will look
slimy. To completely dissolve the powder, put the solution at
À20
C and stir every 30 min until it almost freezes, then put it
rocking at 4
C for 2 days. To remove any undissolved powder,
centrifuge the solution at 13,200 RPM for 30 min, this should
make the solution clear. Store at À20
C.
6. Custom-made casting mould (Fig. 6).
88
Zain Alhashem et al.
For all solutions, ultrapure sterile water is used (prepared by purifying deionized water, 18 MΩ-cm at 25
C, and autoclaving). Store
all solutions at room temperature unless otherwise indicated.
2.1 Microscopy
Setup
1. Upright compound microscope with a 20Â or 40Â high
numerical aperture dipping lens (see Note 1).
2. Having a motorized stage will allow imaging several embryos in
every session greatly enhancing experimentation efficiency.
This is especially important, when working with genetic condition in which not all specimens are carriers. Having said that, a
motorized stage is not strictly required.
3. Temperature regulation. This can be obtained by having a
temperature chamber enclosing the microscope head, or by
having a tight regulation of the temperature of the room. In
either case, make sure to stabilize temperature of all the components for at least 1 h before initiating imaging, and that the
temperature is the same at every imaging session.
4. Confocal or spinning disk heads. The use of a spinning disk
head will greatly reduce the time of imaging, allowing to image
a greater number of embryos per session and reducing phototoxicity.
2.2 Mounting
Embryos and Imaging
1. E3 fish medium. Prepare a 60Â stock solution (1 L): 17.2 g of
NaCl, 0.76 g of KCl, 2.9 g of CaCl 2 ·2H 2 O, 4.9 g of MgSO 4 ·7H 2 O. Adjust volume to 1 L of water. Dilute to a 1Â working
solution as needed.
2. Tricaine (MS-222, Sigma A5040-100G). Prepare a 15 mM
stock solution: Add 0.2 g of tricaine powder and 1 mL of
Tris–HCl 1 M pH 9.5 to 49 mL dH 2 O. Adjust pH to 7 and
store 1 mL aliquots at À20
C. After thawing, tricaine aliquots
can be stored at 4
C for a few days (no more than 1 week).
3. 1% Agarose in 1Â E3.
4. 0.6% Low Melting Point (LMP) agarose in 1Â E3 medium.
5. 3% Methyl cellulose (Sigma M-0387, Viscosity 1500 cPs).
Heat 100 mL 1Â E3 in a closed bottle to 60
C, add 3 g methyl
cellulose and mix with a magnetic stirrer. The solution will take
a long time (overnight or longer) to dissolve and will look
slimy. To completely dissolve the powder, put the solution at
À20
C and stir every 30 min until it almost freezes, then put it
rocking at 4
C for 2 days. To remove any undissolved powder,
centrifuge the solution at 13,200 RPM for 30 min, this should
make the solution clear. Store at À20
C.
6. Custom-made casting mould (Fig. 6).
88
Zain Alhashem et al.