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(modified from a zebrafish protocol provided by Sharon Amacher)
- 1) Fixation
Fix embryos in 4% paraformaldehyde in PBS for 1 hr at RT.
Wash with PBS and dechorionate in PBS.
Wash 1X with MeOH : PBS (1:1), then 2X with 100% MeOH, then incubate in MeOH at RT for at least 1hr.
Store in 100%MeOH at 4oC
- 2) Rehydrate embryos
Wash with MeOH : PBS = 60:40, 30:70, then twice in 100%PBS
Transfer embryos into to desired number of in situ tubes.
- 3) Pre-Hybridization
Wash with PBT 3 times
Wash with PBT : PreHYB=1:1
Wash with PreHYB 2 times
Incubate embryos in pre-HYB at 66oC (0r at optimal temp.), overnight (15-20hrs)
- Pre-Hybridization Buffer:
25ml formamide 50% final
12.5ml 20XSSC 5X final
250 ul 10mg/ml heparin 50 mg/ml final
2100 ul 11.9mg/ml tRNA 500 mg/ml final
500 ul 10% tween-20 0.1% final
460 ul 1M citric acid
sterile water to 50 ml final pH to 6.0
- 4) Hybridization
Dilute probe in PreHYB to 1:50 dilution.
Denature at 95oC for 10 min.
Reduce probe temperature to hybridization temperature (66oC)
Add probe to embryos in tubes
Incubate at 66oC overnight (24-30hrs)
- 5) Washes (Warm solutions to appropriate temp before use)
1 quick wash with HYB
1X5min HYB:2XSSC (2:1), 66oC
1X5min HYB:2XSSC (1:1), 66oC
1X5min HYB:2XSSC (1:2), 66oC
1X5min 2XSSC, 66oC
2X20min 0.2XSSC, 66oC
2X20min 0.1XSSC, 66oC
1X5min 0.1XSSC:PBT (2:1), RT
1X5min 0.1XSSC:PBT (1:1), RT
1X5min 0.1XSSC:PBT (1:2), RT
1X5min PBT, RT
- 6) Transfer embryos to new tubes
- 7) Block
Rinse 2X in blocking solution
Incubate 1-2hr in blocking solution at RT
Blocking solution:
2% sheep serum and 2 mg/ml BSA in PBT
(1ml sheep serum + 100mg BSA + PBT to a final volume of 50 ml)
- 8) Anti-DIG antibody incubation
Incubate in Anti-DIG Fab (1:5000, 1ml FAB in 5ml blocking solution ) at 4oC overnight (with gentle rocking) then 4 hrs at RT (with gentle rocking)
- 9) Washes
All washes are at RT.
2X quick washes in PBT
3X15min in PBT
2-3 X 1hr in PBT
2X quick washes in Coloration Buffer
2X5min in Coloration Buffer
Coloration Buffer:
5 ml 1M Tris-HCl, pH 9.5 100mM final
2.5 ml 1M MgCl2 50mM final
1 ml 5M NaCl 100mM final
5 ml 10% Tween-20 0.1% final
sterile water to 50 ml
- 10) Transfer embryos to clear tubes
- 11) Color reaction
Add color reaction solution to embryos (dissolve half of one NBT/BCIP tablet in 5ml water, supplemented with NaCl to a final conc. of 100mM)
Incubate embryos at RT in the dark until desired intensity is reached (can incubate O/N at 4C if desired)
- 12) Washes
All washes are at RT
Wash several times in PBS
Dehydrate through an EtOH series: 50%, 80%, 90%, 95%, 100%, 100% (5min each)
Wash once in 100% PPO, 1min
Aloow to stand in PPO : EPON (1:1), for ~ 45 min (NOTE: PPO is toxic, handle carefully)
Allow PPO to evaporate (~45 min in hood, then ~45 min under vacuum)
Transfer to fresh Epon
Store Epon-cleared embryos at 4oC