III. HYBRIDIZATION IN 96 WELL PLATES
1. Prepare fixed embryos as described in section I.
2. Add 4 ml hybridization buffer WITHOUT dextran sulfate to 1.5ml of settled embryos.
3. Rock embryos for at least 1 hr at RT.
4. Using a multichannel pipettor with cut-off yellow tips, add 20μlembryos to each well of a 96 well filtration plate (Millipore MADV N65).
5. In a separate (non-filter) 96 well plate, mix in each well 200μlhybridization buffer
WITH dextran sulfate and 5μlof probe.
6. Using multichannel pipettor, add probes to wells of filtration plate.
7. Seal plate with tape and rock at 55 degrees C overnight.
8. Carefully remove cover, then add 100μlwarm wash buffer and place plate on vacuum manifold (Millipore MAVM 096 01).
9. Turn vacuum to LOWEST setting, press on top of plate to form seal. Once the last bit of hybridization buffer has been removed from wells quickly turn vacuum off.
(Make sure vacuum is set to lowest setting. If the vacuum is too high the embryos will become flattened and stick to the membrane.)
10. Empty the manifold tray containing hybridization buffer.
11. Use a multichannel pipettor to add 200μlwash buffer to each well, then remove
wash buffer with LOW vacuum.
12. Repeat steps 10 and 11.
13. Add 200μlwash buffer to each well, then rock 1hr. at 55 degrees C.
14. Remove wash under LOW vacuum.
15. Repeat steps 13 and 14 another 6 times.
16. Add 200μlwash buffer, seal plate, then rock overnight at 55 degrees C.
17. Rinse embryos with 200μlPBT.
18. Add 200μlPBT, rock 30 min. at RT.
19. Remove PBT, add 200μlPBT + 5% Normal Goat Serum + anti-digoxigenin Alkaline Phosphatase (1:2000 dilution), rock 2 hrs at RT.
20. Rinse twice with PBT .
21. Wash embryos 9 X for 10 min. in PBT.
22. Rinse twice with 200μlAlkaline Phosphatase buffer (AP buffer).
23. Wash 5 min at RT with AP buffer.
24. Add 200μlAP buffer containing Nitro Blue Tetrazolium (NBT) and Bromo-Chloro- Indolyl-Phosphatase (BCIP).
25. Incubate with rocking until desired color development is achieved (20 min. to overnight). To stop development of individual wells, remove staining solution and add 200μlPBT.
26. To stop entire plate, rinse plate 3X with PBT.
27. Add 200μl70% gycerol to each well.
28. Embryos are ready to mount or dissect once they have settled to the bottom of the well.