- After the ovarioles are positioned, blot away the cultivating
medium using the Whatman filter strips. This will also remove
all the egg chambers that are not fixed under the oil. The
Voltalef 10S oil will subsequently invade the space of the culti-
vating medium. With some filter strips, smear the edges of the
oil to ensure isolation of the selected ovarioles from the atmo-
sphere. Also, blot away the two other drops on the coverslip
fromsteps 8and 9. - Under the oil, arrange the ovarioles as such that the appropri-
ate egg chambers are physically accessible for the microinjec-
tion needle (Fig.1a) - Place and secure the coverslip onto the incised plastic slide
(Fig.1b). - To transfer the specimen to the injection microscope, first
retract the needle from the storing oil droplet. Carefully place
the specimen holder onto the stage and return the needle until
its tip is in the oil droplet that covers the ovarioles (seeNote
10 ). - Check if the microinjection settings are correct and adjust if
necessary (step 6). - Focus the specimen and adjust the tip position to bring it into
focus. - Slowly approach an egg chamber and try to move the tip into
it. A successful piercing is indicated by initial distortion and
sudden relaxation of the surface of the egg chamber (seeNote
13 ). - Once the tip is inside the oocyte, inject with the determined
pressure. Look for the injection effect. Ideally, the organelles
(e.g., yolk granules) should be slightly displaced by the injec-
tion volume and fluorescence of the probes should be detected
(seeNote 14). - Either focus on another egg chamber and repeatsteps 18and
19 or proceed with microscopy.
3.9 Imaging the
Probe Labeled
Transcripts
- Transfer the specimen to the injection microscope and/or
switch to the high magnification imaging objective. - Set up the detection settings as follows (seeNote 15):
(a) 488 nm or514 nm laser excitation, 525 nm–575 nm
detection window in case of TO labeled probes (Fig.2).
(b) 561 nm or594 nm excitation, 605 nm–655 nm detection
window in case of QB labeled probes. - We recommend acquiring images with a minimum of twofold
oversampling in the lateral dimensions (x and y) and with a
temporal resolution where the imaged mRNPs do not change
their position by more than a particle diameter between
In vivo mRNP Detection by FIT Probes 283