1.2.3. 96- well Plate RNA in situ Hybridization
1. Master Embryo Mix (see section 3.2.3.1 note 26).
2. RNA Probe (see section 3.1.4).
3. Genetix Q-Fill 2 plate filler.
4. Millipore MAVM 096 01 vacuum manifold.
5. 300µ l/well 96-well plate (E&K Scientific).
6. Speedball roller (E&K Scientific).
7. Model 1545 Incubator (VWR).
8. Formaldehyde (Sigma).
9. DEPC treated H2O (2.1.3.5).
10. PBS (Dissolve 8 g of NaCl, 0.2 g KCl, 0.24 g KH2PO4 and 2.72 g Na2HPO4 ·7 H2O in 800 ml of DEPC treated H20, adjust pH to 7.4 with HCl, adjust volume to 1L, sterilize by autoclaving).
11. Tween 20 (polyoxyethykenesorbitan monolaurate, Sigma).
12. Methanol (EM Science).
13. PBT (0.1% Tween 20 in PBS).
14. 20X SSC (Dissolve 175.3 g NaCl and 88.2 g of Sodium Citrate in 800 ml of DEPC treated H20, adjust pH to 7, adjust volume to 1L, sterilize by autoclaving).
15. Hybridization buffer (50% formamide, 4X SSC, and 0.01% Tween 20, store in the dark at -20 ℃).
16. Clay Adams¨ Brand Nutator.
17. 50% Dextran Sulfate (Dissolve 25 g of dextran sulfate in DEPC H2O, adjust volume to 50 ml).
18. Hybridization Buffer with dextran sulfate, (50% formamide, 4X SSC, 5%dextran sulfate and 0.01% Tween 20 store in the dark at -20 ℃).
19. Gyrotory¨ shaker Model G2 (New Brunswick).
20. Wide opening pipete tips (Rainin).
21. 25 ml reagent reservoir (Matrix).
22. 96-well filter plate (Millipore MADV N65)
23. Wash buffer (50% formamide, 2X SSC and 0.01% Tween 20; prepare fresh before use).
24. Goat Serum (Roche, store at -20℃).
25. Anti-Digoxigenin-AP Fab Fragments (store at 4 ° C) (Roche).
26. AP Buffer (0.1M NaCl, 0.05M MgCl2, 0.1M Tris pH 9.5, 0.1% Tween 20; prepare fresh before use).
27. Developing solution (Roche, 45 µl NBT and 35 µl BCIP, store at -20℃, per 10 ml of AP Buffer; add NBT/BCIP just before use).
28. Absolute ethanol.
29. 70% Glycerol (Sigma, 70:30 Glycerol:PBS, filter sterilize).
30. Aluminum plate sealers (E&K Scientific).
31. Low magnification microscope (Zeiss-Stemi 2000-C).
2. Methods
2. Add 1.0 ml of Carbenicillin /SB mix into each well of a 96-well Ritter Riplate using a multichannel pipette.
3. Add 10 µl of thawed cells per well using a multichannel pipette (Brand).
4. Seal tightly with Airpore Tape Sheets.
5. Place Ritter Riplate in a 37 ° C shaking incubator set at 300 RPM for 18 hrs.
6. Store at 4 ° C.
7. Cells are now ready for PCR.
2.1.2. PCR
1. Dilute cells 1:100 in sterile deionized H2O.
