2.2.2. Mass Embryo Fixation
Keep embryos of different time periods separate during fixation
1. Collect embryos from food tray by rinsing the tray with deionized water and capturing the embryos in the three level sieve. The sieving removes fly food and yeast paste17.
2. Fill reservoir18 with 100% bleach and shake embryos for 3 min.19 to remove the chorion membrane.
3. Wash well with distilled water.
4. Remove embryos form the sieve using a spatula and place in a 50ml falcon tube. Fix embryos by gently shaking in 50-50 mix of heptane20 and 4% formaldehyde/PBS21 fixative for 25 min.
5. Remove lower aqueous phase and replace with equal volume of methanol.
6. Shake for 1 min then allow embryos to settle. DO NOT VORTEX.
7. Remove upper phase22 containing the vitelline membranes and embryos remaining at interphase23.
8. Remove remaining methanol24.
9. Wash 3x in methanol.
10. Top off 50ml Falcon tube with methanol.
11. Store embryos at -20 ℃25.
12. Embryos are now ready to be hybridized.
2.2.3. 96-well Plate RNA in situ Hybridization
1. Prepare a master embryo mix by combining equal amounts of embryos from all six time periods in a 50 ml Falcon tube26.
2. Use 1ml of embryos from the master mix per 96-well plate and place them in a 15 ml Falcon tube.
3. Rehydrate in 3:1 methanol:2.5% formaldehyde in 1X PBS 27, 28 for 2 min.
4. Rehydrate in 1:3 methanol:2.5% formaldehyde in 1X PBS29 for 5 min.
5. Post-fix in 2.5% formaldehyde in 1X PBS for 10 min 30.
6. Rinse 6x in PBT.
7. Add 3 ml of hybridization buffer without dextran sulfate per 1 ml of embryos31
8. Incubate with shaking at 125 rpm on the Gyrotory¨ shaker for at least 1 hr at room temperature to pre-hybridize embryos.
9. During pre-hybridization put 200 µl of hybridization buffer with dextran sulfate into each well of a 96-well plate using multichannel pipette.
10. Add 2 µl of probe into each well in columns 1-11. Add 2 µl of control probe into wells in column 12. (Use one well in column 12 for a negative control and do not add probe).
11. Mix thoroughly on a vortex mixer at maximum speed for 25 sec and centrifuge at 4000 rpm for one minute.
12. Carefully pour pre-hybridized embryos into a 25 ml reagent reservoir.
13. Add 20 µl of embryos into each well of a 96-well filter plate (using a multichannel pipette with wide opening tips) 32.
14. Transfer the probes from the 96-well plate into the 96-well filter plate and seal the filter plate with an aluminum foil sealer.
15. Incubate at 55 ℃ with shaking at 125 rpm on the Gyrotory¨ shaker overnight 33.
16. Add 100 µl of room temperature wash buffer.
17. Remove the hybridization-buffer, wash-buffer mix using vacuum; once all the liquid is removed from the wells quickly turn off the vacuum34.
18. Rinse 2x with wash buffer.
19. Incubate in wash buffer at 55 ° C with shaking for 30 min with eight changes.
20. Incubate in wash buffer at 55 ℃ with shaking overnight.
21. Rinse in PBT.
22. Incubate in PBT at RT with shaking for 30 min; remove PBT.
23. Incubate in PBT, 5% goat serum, 1:2000 dilution Anti-Digoxigenin-AP Fab Fragments at RT with shaking for 2 hrs.
