- The brightness is calculated as follows:
Br¼QYεðÞ 485 = 560
1000- The probes with the best fluorescence properties (brightness
and responsiveness) are tested by wash-free FISH (Subhead-
ing 3.6) and subsequently may be used for in vivo
visualization.
3.5 Fly Dissection 1. Take a clean 3–9 well dissection plate, fill one well with 100%
ethanol (~2 mL) and two wells with BRB80.
- Anesthetize the females prepared for dissection (seeNote 3)
and transfer them into the well containing the ethanol, make
sure they sink to the bottom (seeNote 4). - After 30 s incubation, transfer them to the next well with
BRB80 and wash away the ethanol. Transfer them to the
third well. - Using the #4 forceps crush the thorax of the fly and hold it
steady. With the #5 forceps gently grab the ventral side of the
abdomen close to the posterior tip and with a firm movement
open it. - Isolate the bulging ovaries and separate them from the rest of
the internal organs (e.g., intestines, Malpighian tubules, and
the oviduct). - Carry on with the wash-free FISH or the microinjection
protocols.
3.6 Wash-Free FISH To test whether the probes have access to the target RNA in its
native context and conformation and that the RNA–probe duplex
yields sufficient signal–noise ratio a rapid wash-free FISH is carried
out.
- Transfer the dissected ovaries into 500μL fixative in a 1.5 mL
Eppendorf tube and nutate them for 20 min at room tempera-
ture (seeNote 5). - Remove the fixative and wash with 1 mL IBEX three times
10 min. - Add 500μL fresh IBEX prewarmed to 37C and place the
ovaries into the heating block (seeNote 6). - Add the probe(s) to be tested at a 10–50 nM per probe
concentration. - Rock for 20 min with 1200–1400 RPM at 37C.
- Take the tube out of the heating block and allow the ovaries to
settle at room temperature.
280 Jasmine Chamiolo et al.