Lab On-line Methods
|Note: Although it is possible to perform in situ hybridization on bleached embryos, it appears to reduced the strength of the signal. For best results, use albino females.|
- 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.
1. Fix embryos
in 3.7% formaldehyde MEMFA for at least 4 h at r.t.
2. Embryo pretreatment
embryos in 1ml of 10µg/ml proteinase K for 15 minutes
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
6. Wash 2X with PBS-Tween - 5 minutes each
7. Re-fix embryos
for 20 minutes in 3.7% formaldehyde MEMFA,
8. Remove all
but 1ml PBS-Tween and add 250 µl hybridization solution.
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.
hybridization solution with 1ml probe solution (1µg/ml probe).
probe solution and divide into 0.25 ml aliquots, store at -80°C.
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.
probe solution with hybridization solution,
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.
with MAB+block+1:2000 dilution anti-digoxygenin-alkaline phosphotase antibody
HRP-coupled antibodies can also be used, but you have to switch visualization
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.
1. Wash embryos 2X in alkaline phosphatase buffer (or TBS for HRP) - 5 minutes each.
last wash with AP reaction buffer
3. Specimens can be stored in 100% methanol at -20°C or placed in BABB to be cleared.
specimens can be sectioned in wax (which retains the AP staining),
|Table of Methods|