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(Source: S. Schulte-Merker from Zebrafish Book 5th Edition)

Fixation

1. Fix embryos with 4% paraformaldehyde in PBS overnight at 4°C.

2. Wash twice in PBS, 5 minutes each, at room temperature.

3. Remove the embryos from their chorions using watchmaker forceps (easiest at this point).

4. Transfer embryos to vials with 100% methanol (MeOH), replace with fresh methanol after 5 minutes. Use MeOH and not ethanol, because ethanol causes a higher background!

NOTE: From this point on, the embryos remain in the same glass vial until they are ready for prehybridization.

5. Cool the embryos to -20°C for at least 30 minutes (embryos can be stored that way for months; this step is necessary for permeabilization of embryos even if you don't want to store them).

6. Bring the embryos back to room temperature.

7. Immerse 5 minutes in 50% MeOH in PBST and then 5 minutes in 30% MeOH in PBST.

8. Rinse twice in PBST for 5 minutes each.

NOTE: Embryos can also be removed from their chorions at this point, but chorions are sticky after having been in MeOH

9. Fix for 20 minutes in 4% paraformaldehyde in PBS at room temperature.

10. Rinse twice in PBST for 5 minutes each.

Proteinase digestion and postfixation

1. Digest with proteinase K (10 μg/ml in PBST) at room temperature for 5 to 12 minutes (depends on the stage; younger stages are more sensitive; depends, also, on the batch of enzyme; we test each new batch).

2. Rinse briefly in PBST; wash for 5 minutes in PBST.

3. Fix as above (4% paraformaldehyde in PBS, 20 minutes).

4. Wash as above (2 times in PBST).

Acetic anhydride treatment (optional)

1. Replace PBST with dH2O (as quantitatively as possible).

2. As quickly as possible, replace H2O with a fresh mixture of 2.5 μl acetic anhydride in 1 ml of 0.1M triethanolamine (pH 7.0).

3. Incubate for 10-60 minutes at room temperature.

4. Wash 2 times for 10 minutes each in PBST.

This treatment helps reduce background from endogenous phosphatases and is therefore only necessary in cases where background is a problem. It is also unnecessary if peroxidase-coupled anti-digoxigenin antibodies are used.

Prehybridization

1. Transfer embryos (up to 40) into small Eppendorf tubes (0.8ml) in approximately 300 μl of HYB*.

2. Incubate 5 minutes at 55ºC; afterwards, replace HYB* with an equal volume of HYB+.

3. Prehybridize at 55ºC for 1-48 hours in HYB+.

Probes
Prepare RNA Probes according to the Boehringer instructions (Cat. #1175025). About 5 to 10 μg of digoxigenin-labeled probe is transcribed from 1 μg of a linearized plasmid. Hydrolyze the probes to an average length of 150-300 nucleotides following the protocol of Cox et al. (1984; Dev. Biol. 101:485-502). After the final precipitation, the hydrolyzed probe should be taken up directly in HYB+ and stored at 20ºC.

Hybridization

1. Remove as much of the preHYB+ as possible without letting the embryos touch air.

2. Add 20 to 40 μl of fresh HYB+ containing 20-100 ng of RNA probe (about 0.5-5.0 ng/μl) so that all embryos are covered by the solution. Heat the probe in HYB+ for 5 minutes at 68ºC before adding to the embryos. For some probes, signal intensity decreases below 10 ng; amounts higher than 100 ng do not usually give a higher signal intensity. Probably the amount of probe to add depends upon the amount of RNA you want to detect and has to be titrated.

3. Incubate overnight at 55°C.

Probe removal

Remove probe (probes can be reused twice; using them more often results in weaker signals. The probes in HYB+ are stable for at least half a year).

Option A:

1. Soak embryos for 20 minutes at 55°C in 50% formamide in 2xSSCT.

2. Rinse 3 times for 10 minutes each at 37°C in 2xSSCT.

3. Rinse for 5 minutes at 37°C in PBST.

4. Digest for 30 minutes at 37°C in RNAse (RNAse A, 20 μl/ml plus RNAse T1, 100 U/ml in PBST).

5. Rinse 10 minutes at 37°C in 2xSSCT.

6. Soak 60 minutes at 55°C in 50% formamide in 2xSSCT.

7. Rinse 15 minutes at 55°C in 2xSSCT.

8. Rinse twice for 15 minutes each at 50°C in 0.2xSSCT.

9. Rinse 5 minutes in PBST at room temperature.

10 Transfer embryos to microwell dish.

Option B:

1. Soak embryos 2 times for 30 minutes each at 55°C in 50% formamide in 2xSSCT.

2. Rinse 15 minutes at 55°C in 2xSSCT.

3. Rinse 2 times for 30 minutes each at 55°C in 0.2xSSCT.

4. Transfer embryos to microwell dish.

IMPORTANT: For some probes, the RNAse treatment is unnecessary and decreases the signal intensity. For other probes, omitting the RNAse leads to an unacceptably high background. It is therefore advisable to test whether the RNAse treatment is necessary for any given probe. If you can do without it, follow Option B; if not, use Option A.

Detection

1. Block for 1 hour at room temperature with PBST plus blocking reagent (skimmed milk, new born calf serum, BSA, etc.; the Fab fragments are not very sticky, so it doesn't seem to matter what one uses).

2. Add Fab-AP as supplied by Boehringer at a 1:4000-8000 dilution and shake for 4 hours at room temperature in PBST plus blocking reagent.

3. Wash 4 times for 25 minutes each with PBST plus blocking reagent.

4. Wash 3 times for 5 minutes each in staining buffer.

5. Incubate in staining buffer with 4.5 μl NBT and 3.5 μl X-Phosphate (NBT, 75 mg/ml in 70% dimethylformamide; X-Phosphate, 50 mg/ml in dimethylformamide) per ml added.

6. Stain for 30 minutes to overnight.

7. Wash in PBS.

8. Dehydrate with 100% MeOH twice (10 minutes each) and mount in a 2:1 mixture of benzylbenzoate:benzylalcohol. This mixture has the same refractive index as yolk and clears the embryos very well.

If you use alkaline phosphatase as a detection enzyme you need to be aware that NBT/X-Phosphate will fade in anhydrous solutions. Fixing the embryos after the color reaction is necessary if you want to clear the embryos in alcohol. After fixation (4% paraformaldehyde at room temperature for at least half an hour), even weaker signals are reasonably stable in alcohol. You can also overstrain with careful monitoring of the dehydration and clearing process. Another alternative is to photograph weak signals immediately after transferring the specimen to alcohol or to clear them in glycerol.

Solutions:

In situ hybridization

PBST PBS plus 0.1% Tween

SSCT SSC plus 0.1% Tween

HYB* 50% formamide
5xSSC
0.1% Tween-20

HYB+ HYB* with
5 mg/ml torula (yeast) RNA
50 μg/ml heparin

The torula RNA is prepared by proteinase K digestion of RNA with subsequent phenol-, phenol-chloroform-, and chloroform-extraction. The RNA is precipitated and dissolved in DEPC-treated water. HYB* and HYB+ should be kept at -20°C.

In situ Hybridization staining buffer
100 mM Tris pH 9.5
50 mM MgCl2
100 mM NaCl
0.1% Tween-20
1 mM Levamisol (add fresh)