3.2 Imaging Plate
Preparation
1. Heat a water bath to 50
C. Collect 25 mL of thin albumen
from the eggs incubated overnight in a sterile 50 mL tube and
stir it for 15 min at RT (see Note 5).
2. Add 0.75 mL of 10% glucose to the albumen and place the
mixture in the water bath.
3. Boil 25 mL of 0.6% BactAgar in MilliQ H 2 O and add
0.615 mL of 5 M NaCl. Transfer this solution into another
sterile 50 mL tube and place it into the water bath to equilibrate to 50
C.
4. Mix the solutions of Albumen/Glucose and Agar/NaCl in a
sterile 50 mL tube and add 100 μL of P/S (see Note 6).
5. Use a sterile Pasteur pipette to pour 1.5 mL of the Albumen/
Agar mix onto the cell culture inserts on a flat surface (see Note
7), leaving the Albumen/Agar imaging plates at RT to cool
until the gel becomes solid and then storing them at 4
C (see
Note 8).
3.3 Mounting and In
Vivo Time-Lapse
Imaging
1. Reopen the tape-sealed window in the egg, and carefully
remove the thick albumen surrounding and covering the
embryo with a soft tissue (see Note 9).
2. Place a paper ring on top of the vitelline membrane so that the
embryo is located in the center of the clover-shaped hole
(Fig. 3a, b), and cut through the vitelline membrane and
around the whole perimeter of the filter paper ring with a
small pair of scissors, carefully pulling the filter with the embryo
attached away from the yolk (see Note 10).
3. Place the embryo ventral side up in a petri dish containing 1Â
PBS, and clean the remaining yolk and debris by blowing
streams of PBS over the embryo with a Pasteur pipette (see
Note 11).
4. Select the embryos with the best overall morphology and the
greatest level of transgene expression for imaging (see Note
12).
5. Transfer the selected embryos to an Agar/Albumen imaging
plate dorsal side up (Fig. 3c), and fill each well of the culture
chamber with 1.5 mL of a solution of 5 mL thin albumen and
5 mL of 123 mM NaCl.
6. Transfer the embryos in the imaging plates to the wells of the
culture chamber (see Note 13) and add 1Â PBS between the
wells to maintain a moist environment inside the culture chamber. Seal the culture chamber with electrical insulation tape so
that up to six embryos can be imaged at the same time (Fig. 3d,
e: see Note 14).
7. Visualize the embryos under an upright wide-field microscope
Axio Imager 2 (Zeiss) equipped with a motorized stage and an
192
Elena Gonzalez-Gobartt et al.
Preparation
1. Heat a water bath to 50
C. Collect 25 mL of thin albumen
from the eggs incubated overnight in a sterile 50 mL tube and
stir it for 15 min at RT (see Note 5).
2. Add 0.75 mL of 10% glucose to the albumen and place the
mixture in the water bath.
3. Boil 25 mL of 0.6% BactAgar in MilliQ H 2 O and add
0.615 mL of 5 M NaCl. Transfer this solution into another
sterile 50 mL tube and place it into the water bath to equilibrate to 50
C.
4. Mix the solutions of Albumen/Glucose and Agar/NaCl in a
sterile 50 mL tube and add 100 μL of P/S (see Note 6).
5. Use a sterile Pasteur pipette to pour 1.5 mL of the Albumen/
Agar mix onto the cell culture inserts on a flat surface (see Note
7), leaving the Albumen/Agar imaging plates at RT to cool
until the gel becomes solid and then storing them at 4
C (see
Note 8).
3.3 Mounting and In
Vivo Time-Lapse
Imaging
1. Reopen the tape-sealed window in the egg, and carefully
remove the thick albumen surrounding and covering the
embryo with a soft tissue (see Note 9).
2. Place a paper ring on top of the vitelline membrane so that the
embryo is located in the center of the clover-shaped hole
(Fig. 3a, b), and cut through the vitelline membrane and
around the whole perimeter of the filter paper ring with a
small pair of scissors, carefully pulling the filter with the embryo
attached away from the yolk (see Note 10).
3. Place the embryo ventral side up in a petri dish containing 1Â
PBS, and clean the remaining yolk and debris by blowing
streams of PBS over the embryo with a Pasteur pipette (see
Note 11).
4. Select the embryos with the best overall morphology and the
greatest level of transgene expression for imaging (see Note
12).
5. Transfer the selected embryos to an Agar/Albumen imaging
plate dorsal side up (Fig. 3c), and fill each well of the culture
chamber with 1.5 mL of a solution of 5 mL thin albumen and
5 mL of 123 mM NaCl.
6. Transfer the embryos in the imaging plates to the wells of the
culture chamber (see Note 13) and add 1Â PBS between the
wells to maintain a moist environment inside the culture chamber. Seal the culture chamber with electrical insulation tape so
that up to six embryos can be imaged at the same time (Fig. 3d,
e: see Note 14).
7. Visualize the embryos under an upright wide-field microscope
Axio Imager 2 (Zeiss) equipped with a motorized stage and an
192
Elena Gonzalez-Gobartt et al.