HEAT: RAMP + 5 C, PULL¼35, VEL¼75, TIME¼130 d,
PRESSURE¼ 500- Insert a new capillary and execute the program.
- Repeatstep 3to obtain at least 8–10 microinjection capillaries.
3.8 Microinjection
and Imaging
- Prepare the probe(s) for injection by diluting to 2–10μM/
probe final concentration in injection buffer. - Spin down at min 12,000gfor 2 min to remove any aggre-
gates or impurities that may clog the microinjection needle.
Transfer the supernatant to a clean tube. - Load 1.5–2.5μL of the prepared mixture into a microinjection
needle and mount it into the capillary holder of the injector
system (seeNote 9). - Place a drop of Voltalef 10S oil onto a clear coverslip and focus
it with a low magnification objective on the stage of the injec-
tion microscope. - Using the micromanipulator of the injection system, plunge
the tip of the microinjection needle into the oil (seeNote 10). - Apply some positive pressure to the needle and observe the
flow of the injection solution. Adjust the pressure and duration
of the injection until a small (diameter ~20–50μm) liquid
bubble forms under the oil. Typically, this happens at
1000–1200 hPa applied for 0.2–0.3 s using a gas operated
injector (seeNote 11). - Leave the tip under the oil until preparing the specimen (see
Note 12). - Dissect ovaries as described in Subheading3.5 and transfer
them onto one side of a clean coverslip in a small drop of
BRB80 with the #5 forceps. - Place them into a drop of oocyte cultivating medium (5μL) in
the middle of the same coverslip for 10s. - Transfer them to another drop of cultivating medium close to
the other side of the same coverslip. Place a drop of Voltalef
10S oil adjacent to the specimen that the two drops form an
interface. - Using cleaned tungsten needles, separate the ovarioles from
each other. Isolate those that contain oocytes in the appropri-
ate developmental stage(s) and using the germarium as handle,
pull the ovarioles to the medium–oil interface with one needle.
Move the young egg chambers under the oil and leave the rest
in the medium. Remove the late oogenetic stages from the
ovarioles if you do not need them, as they may hinder injection
of the earlier oocytes (Fig.1a).
282 Jasmine Chamiolo et al.