in situ hybridization

in situ hybridization - Harland , R.M. 1991. Meth. Cell Biol. 36:685. 
The first time you use a particular probe it is worthwhile to preabsorb it. This takes about 24 h and dramatically increases the quality of the final product. 

The embryos you use to pre-absorb probe will be sacrificed to the cause of science and aesthetics, so it is best to use wild type specimens fixed for this purpose.

Note: We use 1.5 ml glass vials with screw caps to hold the specimens during the process.  These vials can be labeled with a diamond tip pen to insure that the labeling does not come off during the process.

Pre-hybridization:

1. Fix embryos in 3.7% formaldehyde MEMFA for at least 4 h at r.t. 
     Rinsed 2X with PBS (10 minutes each wash) and store at -20°C in 100% methanol.

2. Embryo pretreatment / rehydration 
    75% ethanol / 25% PBS-Tween - 5 minutes 
    50% ethanol / 50% PBS-Tween - 5 minutes 
    25% ethanol / 75% PBS-Tween - 5 minutes 
    2X 100% PBS-T - 5 minutes each

3. Incubate embryos in 1ml of 10µg/ml proteinase K for 15 minutes  
    (1 µl of 10mg/ml ProK stock solution in 1 ml PBS).  
   

Depending on the age and size of the embryo. The 15 minute time period may have to be adjusted.

4. Rinse 2 X in 0.1M triethanolamine pH 7-8 (leave final volume at 5ml) - 5 minutes each.

5. Add 12.5 µl acetic anhydride for every 5 ml of triethanolamine  
   - mix at room temp for 5 minutes and then add a second 12.5 µl aliquot of acetic anhydride 
   - 5 minutes each.

6. Wash 2X with PBS-Tween - 5 minutes each

7. Re-fix embryos for 20 minutes in 3.7% formaldehyde MEMFA,  
    rinse 5 X with PBS-Tween - 5 minutes each.

8. Remove all but 1ml PBS-Tween and add 250 µl hybridization solution.  
   Allow embryos to settle, remove solution and replace with 0.5 ml hybridization solution    - one hour.

Pre-absorb step: done the first time that you use the probe to reduce the amount of background. Once a probe has been pre-absorbed, you can skip to hybridization.

1. Replace hybridization solution with 1ml probe solution (1µg/ml probe).     Hybridize overnight at 62°C, preferably with some mixing.

2. Recover probe solution and divide into 0.25 ml aliquots, store at -80°C.     The specimens can be discarded.

Hybridization:

1. Replace pre-absorb hybridization solution with 1 ml probe solution (0.25 µg/ml probe).  Hybridize overnight at 62°C, preferably with some mixing.

2. Recover probe solution (this can be used many times over), store at -80°C.

3. Replace probe solution with hybridization solution,  
   Incubate at 62°C - 20 minutes.

4. Wash embryos 3X in 2X SSC at 62°C - 20 minutes each.

5. Wash embryos for 2X in 0.2X SSC at 62°C - 30 minutes each (high stringency wash).

6.  Wash 2X in MAB at room temperature - 15 minutes each.

7. Wash in MAB + 3% blocking reagent for 1-2 hours at room temperature with rocking.

8. Replace with MAB+block+1:2000 dilution anti-digoxygenin-alkaline phosphotase antibody   HRP-coupled antibodies can also be used, but you have to switch visualization protocols!  
   

Incubate overnight at 4°C with rocking.

9. Remove the antibody and save, it can be used at least two more times and kept at 4°C for at least two weeks.  
    Wash embryos 5X with MAB for 1 hour each wash with rocking.

Development:

1. Wash embryos 2X in alkaline phosphatase buffer (or TBS for HRP) - 5 minutes each.

2. Replace last wash with AP reaction buffer stop the reaction when it appears dark enough  (10 minutes - 2 hours) by adding 100% ethanol.

3. Specimens can be stored in 100% methanol at -20°C or placed in BABB to be cleared.

4. Stained specimens can be sectioned in wax (which retains the AP staining),  and counter-stained with DAPI or even other antibodies.