Murgantia histrionica embryo In situ hybridization (based on Oncopeltus protocols), Jessica Hernandez
Day 1 (4-5h)
- Warm up some hybridization buffer (1 mL per tube of embryos) in the 60C hyb oven
- Add 1 mL pre-warmed hyb buffer to each tube
- Let embryos incubate in 60°C incubator for 4-6 h. 4 hours is enough
- Prepare probe dilution in hyb buffer (I always start with 1:1000 dilution for any new probe, I am using Alys’s probe protocol)
- Heat the probe to 93C for 3 min, then transfer to ice immediately.
- Remove embryos from incubator and allow them to sit upright for 2 minutes as embryos settle to the bottom. Remove hybridization buffer and add 200 uL of probe
Day 2 (8hrs+)
- Put 2 wash volumes hyb buffer and 1 wash volume 2X SSC (saline-sodium citrate)/0.1% Tween-202 in 60°C incubator to warm up (wash volume = 1 mL/tube of embryos)
- Remove probe.
- Wash in the following solutions:
- 2 x 30 minutes pre-warmed hyb buffer at 60°C from Step 1
- 1 x 30 minutes pre-warmed 2X SSC/0.1% Tween-20 at 60°C from Step 1
- 1 x 30 minutes 2X SSC/0.1% Tween-20 at RT
- 1 x 30 minutes 0.2X SSC/0.1% Tween-20 at RT3
- Rinse 3X in PBST
- To block, wash in the following solution, rocking, at RT:
- 10% sheep serum in PBST for 2 hours
- Add 800 uL of the antibody (anti-Dig-AP or anti-Fluorescein-AP) at 1:1600 dilution in 10% sheep serum in PBST. Incubate at RT for 4 hours, remove anti-dig solution and rinse 3X in PBST, leave overnight in PBST
Day 3
- Wash in 1 mL the following solutions, rocking at RT:
- 5 x 20 minutes in PBST
- 3 x 5 minutes in AP staining buffer4
- Add 500 uL of staining solution5, 6. Cover with aluminum foil and check every hour for staining.
- To stop staining, rinse 3X in PBST followed by 3 x 10 minute washes in PBST at RT.
- If doing a double stain, inactivate the phosphatase by heating embryos for 30 min at 70 C.
Steps 5 and 6 are absolutely necessary if you are going to mount your embryos on slides, but not necessary if you are going to store in PBST at 4C. If you do not do them, the alkaline phosphatase will still be active and able to make colored product.
- If you used NBT/BCIP:
- Wash with 1:1 PBST/MeOH for 5 min.
- Rinse 2X in MeOH
- Rinse 1X in EtOH
- Rinse 2X in MeOH
- Wash 1:1 PBST/MeOH for 5 min
- If you used INT/BCIP:
- Fix embryos in 4% PFA for 30 min
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1 Hybridization buffer recipe (as of 10/26/16):
- 3.75 mL 20X DEPC SSC
- 75 uL heparin (10 mg/mL)
- 37.5 uL yeast tRNA (20 mg/mL)
- 15 uL Tween-20
- 3.75 mL DEPC-treated ddH2O
- In fume hood, add 7.5 mL formamide.
2 2X SSC/0.1% Tween-20
1.5 mL 20X SSC DEPC
15 uL Tween-20
To 15 mL with ddH2O
3 0.2X SSC/0.1% Tween-20
150 uL 20X SSC DEPC
15 uL Tween-20
To 15 mL with ddH2O
4AP staining buffer (pH 9.5)
300 uL 5M NaCl
750 uL 1M MgCl2
1.5 mL 1M Tris-HCl, pH 9.5
15 uL Tween-20
ddH2O to 15 mL
5BCIP/NBT staining solution (for 2 tubes using 500 uL each)
4.5 uL NBT
3.5 uL BCIP
1 mL staining buffer
6BCIP/INT staining solution
7.5 uL of INT/BCIP stock solution
1 mL staining buffer
If overstained:
High Tween Wash (for removing color stain background):
- mL 5M NaCl
6.25 Ml 1M Tris pH 7.5
2.5 mL Tween-20
31.9 mL ddH2O
Remove NBT/BCIP.
Wash 2X in PBST
Wash 1 x 10 min in High Tween Wash
Wash 2X in PBST
Restain if needed.