Whole Mount In Situ Protocol for DIG & Biotin
Whole Mount In Situ Protocol for DIG & Biotin
RNA Double Labeling
J. W. O'Neill and E. Bier
Dept. of Biology, U.C.S.D.: tel. (619) 534 - 0442 (ask for Brian).
I. Probe Synthesis:
* Always use DEPC treated water ** Filter sterilize all solutions
A. 10X DIG-U, NTP Mix: 10mM ea. A,G,CTP (Boehringer # 1277057); 6mM UTP; 4mM DIG-UTP (Boehringer #1209256).
B. 10X Biotin U-NTP Mix: 8.3mM ea. A,G,CTP; 5.6mM UTP; 7mMbiotin- UTP; 7mM Biotin-UTP (Clonetech #5024-1).
C. 2X Carbonate Buffer: 120mM Na2CO3; 80mM NaHCO3; pH to 10.2.
D. Stop Solution: 0.2M NaAc; pH to 6.0 with acetic acid.
Store all solutions at -20ï¿1⁄2C
1. Into RNase free tubes (Intermountain #C-3302-1) add 1ul of: T7 or T3 RNA polymerase (Promega # p2075, p2083), 10X transcription buffer (comes w/ T7, T3), 10X DIG-U or Biotin- U, NTP mix (see above), 100mM DTT (comes w/ T7, T3), and RNase inhibitor (Promega #N2111). Then add 1ug of linearized DNA and adjust volume of reactants to 10ul total. Incubate at 37ï¿1⁄2C for 2 hrs. 2. Add 15ul H2O and 25ul 2X Carbonate Buffer (see above), mix and incubate at 65ï¿1⁄2C for 20 min.
3. Add 50ul Stop Solution (see above), 15ul 4M LiCl, 5ul 20 mg/ml tRNA (phenol / chloroform extracted), and 300ul 100% EtOH.
4. Mix and freeze (-20ï¿1⁄2C) at least 20 min., then spin hard 15 min. in 4ï¿1⁄2C. 5. Wash pellet in 70% ethanol (use DEPC ddH2O).
6. Dry, then dissolve pellet in 75ul hybridization mix (Hybe = 50% Formamide, 5X SSC, 100 ug/ml Heparin, 0.1% Tween20, 100 ug/ml sonicated and boiled salmon sperm DNA)
II. Fixation / Hybridization
7. Remove chorions in 50% bleach for 3 min.
8. Wash with TXN (0.04% Triton-X 100 + 0.7% NaCl), dry excess TXN.
9. Scoop embryos into scintillation vial with Fixation Buffer (4ml PBS w/ 0.05M EGTA and 9.0% Formaldehyde, and 4ml Heptane), shake vigorously, 25 min.
10. Remove aqueous layer (bottom).
11. Add 30ml 100% MeOH, remove heptane layer.
12. Wash 2X with 30ml of 100% EtOH and store at -20ï¿1⁄2C.
13. To 75ul settled embryos, add 500ul xylenes / 500ul ethanol for 30 min.
14. Wash with EtOH 5X, MeOH 2X, then 3X with PBT ( PBS+0.1% Tween). 15. Fix for 25 min. in PBT+ 5% formaldehyde, wash 5X PBT.
16. Incubate for 8 min. in 500ul PBT+ 4 mg/ml non-predigested Proteinase K. 17. Wash 4X with PBT, then add 500ul PBT+ 5% formaldehyde for 25 min. 18. Wash 5X PBT, then add 500ul PBT / 500ul hybridization solution (Hybe, see above) 19. Wash 3X in Hybe, then pre-hybridize for 1 hr in 500ul, 55ï¿1⁄2C. 20. To embryos add 75ul Hybe, then add DIG probe at 1:100 and Biotin probe at 1:60 total volume (Embryos + Hybe = 150ul).
21. Incubate at 55ï¿1⁄2C O/N (12+ hrs).
22. Wash 4X with Hybe over 1.5 hrs at 55ï¿1⁄2C.
**** If doing Biotin probe stain only, go to #29.
III. Staining Reactions:
Anti - digoxigenin (DIG) - Alkaline Phosphatase Reaction:
23. Add 500ul Hybe / 500ul PBT, then wash 4X with PBT over 1hr at room temp.
24. * Refix for 15 min. in PBT+ 5% formaldehyde. Wash 5X PBT, then 3X over 15 min.
25. Use pre-absorbed anti-DIG-AP (Boehringer #1093274) at 1:2000 final in 500ul PBT, 1hr. Wash 4X over 1hr with PBT. Pre-absorb by diluting anti-DIG 1:10 against an equal volume of fixed embryos in PBT, 12hrs at 4ï¿1⁄2C.
26. Wash 3X over 15 min. in Alkaline Phosphatase staining Buffer (APB = 100mM NaCl, 50mM MgCl2, 100mM Tris pH 9.5, and 0.1% Tween20).
27. Transfer embryos to 24 well plates then add 400ul of APB + BCIP ( 0.38mM) and NBT (0.41mM). Blue stain will form in 20 min. to 2hrs (See Boehringer anti-DIG-AP protocol).
28. Stop reaction w/ 6X washes of 95% EtOH. Store in 100% EtOH.
* note: Don't use step #24 if only staining for DIG.
Anti-Biotin / Peroxidase Reaction:
29. (If coming from #22, step with 500ul Hybe / 500ul BSA/PDT.) Wash 5X with BSA/PDT (PBS+ 0.2% BSA, 0.3% Deoxycholate, 0.3% Triton-X 100), then wash 4X over 1hr.
30. Use pre-absorbed anti-Biotin (Boehringer #1297597) at 1:2000 final in 500ul BSA/PDT, 1.5hrs. Wash 6X in BSA/PDT over 1.5hr. Pre-absorb by diluting anti-Biotin 1:10 against an equal volume of fixed embryos in BSA/PDT, 12hrs at 4ï¿1⁄2C.
31. Add an anti-mouse-biotinylated secondary (Vector #BA-2000) at 1:200 for 1hr in 500ul BSA/PDT. Wash 4X in BSA/PDT over 1hr.
32. 30 min. prior to use mix 2.5ul of solutions A and B, from Vector's Elite Vectastain Kit (Vector #pk-6100) to 500ul BSA/PDT. Add 500ul / sample pre- incubated AB to sample for 1hr.
33. Wash 10X over 15 min. with BSA/PDT, then 6X over 10 min. with 0.12M Tris, pH 7.3.
34. Add 500ul of 0.75 mg/ml DAB in 0.12M Tris to embryos, incubate 15 min. Then add 1:1375 30% H2O2 to sample and the brown DAB precipitate will form in seconds to 30 min.
35. Stop peroxidase / DAB reaction with 8 washes of 0.12M Tris, pH 7.3.
36. Step into 100% EtOH (50,70,95%) with several washes and store at 4ï¿1⁄2C. Mount in Permount.
The BIER LAB -- UNIVERSITY of CALIFORNIA, SAN DIEGO Last Updated: 3 November 1997
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