In Situ Hybridization with RNA Probes

Embryo fixation:

1. Remove embryos from molasses plate, wash off yeast, dechorionate in 50% Clorox for 3-5 minutes.
2. Rinse with TXN.
3. Transfer embryos to scintillation vial containing 2.5 ml of ribofix solution and 2.5 ml heptane.
4. Shake at 125 rpm for 25 minutes.
5. Transfer embryos to a labeled 1.7 ml tube, remove all formaldehyde, leave embryos in 0.5 ml heptane.
6. Add 0.5 ml MeOH, and shake vigorously in hand for 30-60 seconds.
7. Rinse twice with MeOH, then twice with 100% EtOH. Store in EtOH at -20°C.

Preparing DNA to make RNA probes

1. Linearize 5 micrograms of DNA in a regular 20 microliter digest, 2 hr at 37°C.
2. Extract with phenol/DEPC HOH:
   
   • add 80 microliters DEPC H2O to digest
   • add 100 microliters phenol
   • rock for 2 minutes
   • spin for 2 minutes at 12K rpm
   • transfer top layer to new tube
3. Extract with high quality chloroform, 100 microliters, rock and spin as above, transfer top layer to new tube.
4. Precipitate DNA by adding 10 microliters 3M NaOAc in DEPC H2O and then 250 microliters cold 100% EtOH (the good stuff). Incubate at -20°C for 2-3 hr.
5. Spin tubes 5-10 minutes in cold room at top speed, wash pellet in 70% EtOH (made with DEPC H2O), repeat spin, air dry about 1 hour.
6. Resuspend pellet in 10 microliters DEPC H2O. From this step on all water added must be DEPC treated, and if possible all solutions made in DEPC water and filter sterilized.

Probe synthesis:

1. In RNAse-free tubes, add 1 microliter each of:
   • T3, T7, or SP6 RNA polymerase
   • Corresponding 10X transcription buffer
   • 10X DIG-U NTP mix (Roche/BMB)
   • 100 mM DTT (if necessary; check your transcription buffer to see if it already contains it.)
   • RNAsin
2. Add 2.5 micrograms of linearized DNA (=5 microliters)
3. Adjust volume to 10 microliters, and incubate at 37°C for 2 hours.
4. Add 15 microliters H2O and 25 microliters 2X Carbonate Buffer, and incubate at 65°C for 20 minutes.
5. Add:
   • 50 microliters Stop Solution
   • 15 microliters 4M LiCl
   • 5 microliters 20 mg/ml yeast tRNA
   • 300 microliters cold 100% EtOH
6. Mix and freeze at -20°C for at least 20 minutes.
7. Spin hard at 4°C for 15 minutes.
8. Wash in 70% EtOH and repeat spin.
9. Dry pellet, then resuspend in 75 microliters hybe solution. Store at -20°C, or for infrequently used probes, -80.

Hybridization:

1. While performing all of these washes begin preadsorbing Ab: wash o/n OreR embryos 2X in MeOH, 1X in 50% MeOH, 7X in PBT, all 5 minutes each, then rock at 4°C overnight with Ab diluted 1:2000 in PBT (no goat serum).
2. To 75 microliters of fixed embryos add 0.5 ml xylenes (organic waste), 0.5 ml EtOH, rock 30 min.
3. Rinse 5X with EtOH, 2X with MeOH, then do 3 washes in 0.1% PBT, rocking for 5 minutes each.
4. Refix for 25 minutes in PBT + 5% formaldehyde, rocking.
5. Wash 5X in .1% PBT in DEPC, 5 minutes each.
6. Perform PK treatment in 0.5 ml .1% PBT in DEPC, with PK at 4 micrograms/ml (=1:5000 from 20 mg/ml stock), rock for 8 minutes.
7. Wash 4X in PBT, 3 minutes each.
8. Refix in PBT + 5% formaldehyde for 25 minutes, rocking.
9. Wash 5X in .1% PBT in DEPC, then 1X in 1:1 PBT:hybe, 5 minutes each.
10. Wash 3X in hybe, prehybridize in 0.5 ml hybe at 55° for 1 hour.
11. Pull off hybe, add 125 microliters hybe and add probes. Use DIG probes at 1:25 (5 microliters). Swirl embryos to disperse probe and incubate at 55°C overnight.
    
12. Wash embryos 4X in hybe, 20 minutes each at 55°C, rocking by hand now and then.
13. Wash in PBT:hybe, then 4X in PBT, 15 min each, at room temperature.
14. Add preadsorbed anti-DIG Ab at 1:2000 in 0.5 ml PBT, rock 1 hour.
15. Wash 4X in PBT , 15 minutes each.
16. Wash 3X in freshly made AP Buffer, 5 minutes each.
17. In 0.5 ml AP Buffer add 1.75 microliters X-Phosphate, 2.25 microliters NBT, rock until color reaction is visible to naked eye, monitor reaction under dissecting scope, stop when good and dark by washing 7X with PBT. Store at 4°C.

Recipes:

TXN:

• 0.04% Triton X-100
• 0.7% NaCl

Ribofix Solution:

• 1.4 ml 16% formaldehyde (EM grade, Polysciences)
• 0.25 ml 10X PBS
• 0.25 ml 0.5M EGTA, pH8
• 0.6 ml H2O

AP Buffer:

• 100 mM NaCl
• 50 mM MgCl2
• 100 mM Tris-HCl, pH 9.5
• 0.1% Tween-20

For buffered phenol in DEPC-H2O, see Maniatis, p. B4.

X-Phosphate, NBT, anti-DIG AP conjugate, and DIG-U NTP mix are all from Roche/BMB.

2X Carbonate Buffer:

• 120 mM Na2CO3
• 80 mM NaHCO3
• pH solution to 10.2 with NaOH

Stop Solution:

Hybe Solution:

• 50% formamide
• 5X SSC
• 100 micrograms/ml heparin
• 0.1% Tween-20
• 100 micrograms/ml sonicated, boiled salmon sperm DNA

Protocol adapted by Jake Harrison from O'Neill and Bier, Biotechniques 17, no. 5, p. 875, 1994.