2.3 Image Analysis
1. TIFF files of a time-lapse stack.
2. High specification PC with the following software: Java 6 or
later (preferentially Java 8), Fiji (Image J), Interactive Data
Language (IDL), MATrix LABoratory (MatLab).
2.4 Tamoxifen
Induction
4-HydroxyTamoxifen (Sigma H6278-10MG, or an equivalent reference from other providers). Prepare a 5 mM stock solution:
10 mg of tamoxifen powder in 5.1 mL ethanol. Aliquot and store
at À20
C. Dilute to a working concentration of 0.5 μM in E3.
2.5 Photoconversion
and Embryo
Dissociation
1. Glass bottom petri dishes (35 mm).
2. FACSMax Cell dissociation solution (Amsbio T200100 or an
equivalent reference from other providers).
3. Heat-inactivated bovine serum.
4. Trypsin-EDTA 0.25% cell culture standard.
5. Cooled tabletop centrifuge.
6. Sterile PBS buffer. Add 8 g of NaCl, 0.2 g of KCl, 1.44 g of
Na 2 HPO 4 , 0.24 g of KH 2 PO 4 to 800 mL of water. Adjust the
pH to 7.4 with HCl. Add water to a total volume of 1 L and
autoclave.
7. PBST, PBS with 0.5% Tween.
3 Methods
3.1 Embryo
Mounting
1. Make an agarose cast using a custom-made 8- or 16-pin plastic
mould (Fig. 6). Melt 1% agarose in E3, wait for it to cool to
approximately 60
C. Add 0.6 mL of fresh tricaine to 5.4 mL of
agarose. Place the plastic mold onto a 60 mm petri dish, pour
agarose making sure no bubbles are trapped around the pins
(see Note 3).
2. Let the agarose set and remove the plastic mould. It should
leave 8 or 16 rounded, and evenly spaced holes of the size of
the embryo yolk.
3. Pre-warm the agarose mould on top of a 40
C heating block.
4. Mix 0.9 mL of melted 0.6% LMP and 0.1 mL of tricaine stock
solution in a 1.5 mL tube and leave in the heating block set at
40
C. Make sure that the temperature does not exceed 42
C
as higher temperatures can kill the embryos.
5. Dechorionate the embryos to be mounted in E3/tricaine
(1 mL tricaine stock solution for 25 mL of E3) using n5
tweezers.
6. As soon as you take the LMP agarose from the heating block, it
will start to set. Hence, you have to be as quick as possible in
90
Zain Alhashem et al.
1. TIFF files of a time-lapse stack.
2. High specification PC with the following software: Java 6 or
later (preferentially Java 8), Fiji (Image J), Interactive Data
Language (IDL), MATrix LABoratory (MatLab).
2.4 Tamoxifen
Induction
4-HydroxyTamoxifen (Sigma H6278-10MG, or an equivalent reference from other providers). Prepare a 5 mM stock solution:
10 mg of tamoxifen powder in 5.1 mL ethanol. Aliquot and store
at À20
C. Dilute to a working concentration of 0.5 μM in E3.
2.5 Photoconversion
and Embryo
Dissociation
1. Glass bottom petri dishes (35 mm).
2. FACSMax Cell dissociation solution (Amsbio T200100 or an
equivalent reference from other providers).
3. Heat-inactivated bovine serum.
4. Trypsin-EDTA 0.25% cell culture standard.
5. Cooled tabletop centrifuge.
6. Sterile PBS buffer. Add 8 g of NaCl, 0.2 g of KCl, 1.44 g of
Na 2 HPO 4 , 0.24 g of KH 2 PO 4 to 800 mL of water. Adjust the
pH to 7.4 with HCl. Add water to a total volume of 1 L and
autoclave.
7. PBST, PBS with 0.5% Tween.
3 Methods
3.1 Embryo
Mounting
1. Make an agarose cast using a custom-made 8- or 16-pin plastic
mould (Fig. 6). Melt 1% agarose in E3, wait for it to cool to
approximately 60
C. Add 0.6 mL of fresh tricaine to 5.4 mL of
agarose. Place the plastic mold onto a 60 mm petri dish, pour
agarose making sure no bubbles are trapped around the pins
(see Note 3).
2. Let the agarose set and remove the plastic mould. It should
leave 8 or 16 rounded, and evenly spaced holes of the size of
the embryo yolk.
3. Pre-warm the agarose mould on top of a 40
C heating block.
4. Mix 0.9 mL of melted 0.6% LMP and 0.1 mL of tricaine stock
solution in a 1.5 mL tube and leave in the heating block set at
40
C. Make sure that the temperature does not exceed 42
C
as higher temperatures can kill the embryos.
5. Dechorionate the embryos to be mounted in E3/tricaine
(1 mL tricaine stock solution for 25 mL of E3) using n5
tweezers.
6. As soon as you take the LMP agarose from the heating block, it
will start to set. Hence, you have to be as quick as possible in
90
Zain Alhashem et al.