2 Materials
2.1 Flies
ubi::E-cad-GFP (Bloomington, 58471 or 58742) flies can be used
as the source of E-cad-GFP (see Note 1). The copy number of Ecad-GFP and endogenous E-cad loci must be equal in the control
and experimental animals, as diffusional coefficients calculated from
recovery curves depend on the total concentration of the tagged
protein [26]. Alternatively, flies with endogenously tagged shg::
E-cad-GFP (Bloomington 60584) can be used. In our experience,
ubiquitously expressed and endogenously tagged E-cad behave
identically in FRAP experiments [27].
2.2 Reagents
and Equipment
1. Apple juice plates for the collection of embryos: 60 mm plates
filled with 10 ml of apple juice agar media (for 50 plates: 15 g
agar, 400 ml distilled water, 100 ml apple juice, 7.5 ml 10%
nipagin in absolute ethanol) and baking yeast.
2. Collection chambers, which tightly fit the apple juice plates and
allow oxygen access to the flies.
3. Embryo preparation: bleach and deionized H 2 O.
4. Filtration nets: using a razor blade, cut a 15 ml Falcon tube at
about the 11.5 ml mark and make a hole of about 1 cm diameter in its lid. Then, cut a nylon net out of a 100 μm pore cell
strainer, and assemble the embryo filtration net by inserting the
net between the lid and tube.
5. Dissection needles.
6. Microscope slides.
7. Heptane glue: incubate a 5 cm length of adhesive tape (Sellotape 1447052 is used due to no toxic effects) with 2 ml
heptane for at least 24 h prior to conducting experiments in a
sealed glass vial.
8. Halocarbon oil 27.
9. A confocal microscope with a 488 laser and sufficiently sensitive
detectors.
10. Software packages: Fiji (https://fiji.sc), Microsoft Excel, and
appropriate statistical packages, e.g., Matlab (https://www.
mathworks.com/products/matlab.html), R (https://www.rproject. org), and GraphPad Prism (https://www.graphpad.
com/scientific-software/prism/).
3 Methods
3.1 Embryo
Collection
1. For synchronized egg collections, set at least 50 virgin female
and 20 male flies to mate for 1 day in a collection chamber with
an apple juice plate attached to the bottom of the chamber at
FRAP to Study the Dynamics of Proteins in vivo
147
2.1 Flies
ubi::E-cad-GFP (Bloomington, 58471 or 58742) flies can be used
as the source of E-cad-GFP (see Note 1). The copy number of Ecad-GFP and endogenous E-cad loci must be equal in the control
and experimental animals, as diffusional coefficients calculated from
recovery curves depend on the total concentration of the tagged
protein [26]. Alternatively, flies with endogenously tagged shg::
E-cad-GFP (Bloomington 60584) can be used. In our experience,
ubiquitously expressed and endogenously tagged E-cad behave
identically in FRAP experiments [27].
2.2 Reagents
and Equipment
1. Apple juice plates for the collection of embryos: 60 mm plates
filled with 10 ml of apple juice agar media (for 50 plates: 15 g
agar, 400 ml distilled water, 100 ml apple juice, 7.5 ml 10%
nipagin in absolute ethanol) and baking yeast.
2. Collection chambers, which tightly fit the apple juice plates and
allow oxygen access to the flies.
3. Embryo preparation: bleach and deionized H 2 O.
4. Filtration nets: using a razor blade, cut a 15 ml Falcon tube at
about the 11.5 ml mark and make a hole of about 1 cm diameter in its lid. Then, cut a nylon net out of a 100 μm pore cell
strainer, and assemble the embryo filtration net by inserting the
net between the lid and tube.
5. Dissection needles.
6. Microscope slides.
7. Heptane glue: incubate a 5 cm length of adhesive tape (Sellotape 1447052 is used due to no toxic effects) with 2 ml
heptane for at least 24 h prior to conducting experiments in a
sealed glass vial.
8. Halocarbon oil 27.
9. A confocal microscope with a 488 laser and sufficiently sensitive
detectors.
10. Software packages: Fiji (https://fiji.sc), Microsoft Excel, and
appropriate statistical packages, e.g., Matlab (https://www.
mathworks.com/products/matlab.html), R (https://www.rproject. org), and GraphPad Prism (https://www.graphpad.
com/scientific-software/prism/).
3 Methods
3.1 Embryo
Collection
1. For synchronized egg collections, set at least 50 virgin female
and 20 male flies to mate for 1 day in a collection chamber with
an apple juice plate attached to the bottom of the chamber at
FRAP to Study the Dynamics of Proteins in vivo
147