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specialized equipment and knowledge of how to microinject
oocytes, it is not diffi cult providing the equipment and tools are
available. This protocol is adapted from one fi rst published by
Johnson et al. (2010) [ 20 ].- Prepare a solution of 3 % Ficoll in 0.4× MMR ( see Note 23 ).
- Prepare and calibrate needles of appropriate size for microin-
jection ( see Note 24 ). The cytosolic volume of X. laevis
oocytes is ~500 nL, and this volume should be used to calcu-
late the fi nal intracellular concentration of the injected solu-
tions. The injection volume should be kept below 2 % of the
total egg volume (20 nL in the case of the X. laevis oocytes). - Separate the required number of healthy, stage VI oocytes for
the experiment. - Carefully and slowly coat a clean, glass microscope slide with
the 3 % Ficoll solution. Ensure that none runs over the edges
of the slide. - Transfer the oocytes to be injected to the Ficoll solution
( see Note 25 ). - Inject oocytes with recombinant cytochrome c , to a fi nal con-
centration of 200 nM, and with IRDye 800CW/QC-1 CSP-3
to a fi nal concentration of 200–400 nM ( see Note 26 ). Oocytes
should be injected with the needle at a slight (~30–40°) angle
on the dark half (animal pole) of the oocyte, just above the
border of the dark and light halves. - Transfer injected oocytes to a black 96-well plate (3–5 oocytes
per well), and incubate for 30 min—2 h at room temperature
in OR-2 buffer. - Scan the plate on an Odyssey Infrared Imaging system
(LI-COR), with a focus offset of 3 mm, a resolution of 84 μm,
and an intensity setting of 5 in the 700 nm channel and 0.5 in
the 800 nm channel ( see Note 27 ).
4 Notes
- Frogs can also be housed in dechlorinated tap water. However,
the temperature still needs to be maintained at 18 °C and the
water at an appropriate level of conductivity [ 18 ]. - Ovulated females should have ideally a 3–6-month break in
between cycles of priming and laying. - For our system, injecting frogs with hCG during the after-
noon (~2 pm) results in consistently better quality eggs.
Female frogs will usually begin laying eggs 10–12 h following
injection; we believe the better quality of the eggs results from
less time spent in the tanks prior to collection.
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