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Molecular Methods

CHAPTER 9 - MOLECULAR METHODS

TWO COLOR WHOLE-MOUNT RNA IN SITU HYBRIDIZATION

(Source: G. Hauptmann)

I. Preparation of RNA Probe

Reagents:

1. Add in the following order:

2. Mix and spin down

3. Add:

4. Incubate 2-3 hr at 37C

5. Add 4 ul DNAse and incubate 30 min at 37C

6. Add 40 ul H20DEPC

7. Prepare push column device:

8. Precipitate with 0.5 vol 7.8 M NH4Ac and 3 vol 100% EtOH at -20C for 30 min

9. Wash with 80% EtOH

10. Redissolve in 25 ul H20DEPC

11. Remove 1 ul and analyze on a 1% agarose gel. To check for incorporation you can blot and stain the gel or just do a dot blot.

12. Add 76 ul HB4-H20 and store at -20C

13. First try 1 ul probe in 100 ul HB4 for in situ hybridization

Comments

If you get high background with this probe concentration after a short staining time, dilute your probe. If you get no staining at all, this usually means the probe is not very good. Make a new one.

T7 RNA-Polymerase from Promega usually works and will produce about 10 ug of RNA. In my hands T3 RNA-Polymerase from Promega for some strange reason often gives a very low amount of RNA. But with good templates like krx-20 you still get a very good probe. For difficult templates try T3 RNA-Polymerase from Boehringer which appears to produce more RNA. All in all it is better to use T7 RNA-Polymerase to produce antisense RNA.

Wizard Miniprep DNA can be used to make the templates. Dissolve in H2ODEPC. It is best to use 1 ug of template. More than 2 ug does not increase the amount of RNA significantly and less than 0.5 ug is not very effective.

You can also precipitate with NaAc instead of NH4Ac. If you omit the push column and just precipitate with NH4Ac, to save time you will also get a good separation of nucleotides. However, precipitation with LiCl only is not very effective in removing free nucleotides. Hydrolysis of the probe is not advisable because this reduces the sensitivity of the probes. Probes from 0.5-2.5 kb can be used without hydrolysis. Probes longer than 1 kb work best. If you want to combine different fragments of the same gene to get a stronger probe, mix them before the push column step.

II. Fixation and Storage of Zebrafish Embryos

1. Transfer embryos of the desired stage to a small petri dish.

2. Remove embryos from their chorions using watchmaker forceps.

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

4. Wash 4 x 5 min in PBSTw at RT.

5. Transfer embryos to 100% methanol. Replace with fresh methanol after 5 min. Store embryos at -20C in a 24 well tissue culture plate (Falcon 3047) sealed with parafilm. Embryos should be cooled to -20C for at least 30 min for permeabilization even if you don`t want to store them.

III. Proteinase Digestion and Postfixation

All steps are performed at RT on a shaker.

1. Transfer embryos to a small petri dish.

2. Immerse 5 min in 75% MeOH in PBSTw at RT.

3. Immerse 5 min in 50% MeOH in PBSTw.

4. Immerse 5 min in 25% MeOH in PBSTw.

5. Rinse 2 x 5 min in PBSTw.

6. Digest zebrafish embryos with Proteinase K (10 ug/ml in PBSTw) at RT for 1 to 15 min depending on the stage: 1 cell to high for 1 min; 30% epiboly to 10 somites for 2-3 min; 10-20 somites for 3-4 min 24-32h for 5-6 min; 40-50 h for 10-15 min.

7. Rinse 2 x shortly in 2 mg/ml glycine in PBSTw.

8. Fix in 4% paraformaldehyde in PBSTw for 20 min.

9.Wash 5 x 5 min in PBSTw.

IV. Hybridization

Following steps are performed at 55C for zebrafish.

1. Transfer embryos into 2.0 ml Eppendorf tubes.

2. Prehybridize in 350 ul Hb4 for 1-8 h.

3. Heat the probe in 100 ul Hb4 at 80C for 5-10 min, quickly cool on ice/ethanol, spin down and keep on ice/ethanol. Replace prehybridization solution with probe solution. Incubate overnight.

V. Washes

1. Wash embryos 2 x 30 min in 50% formamide in 2xSSCTw at 55C or at 65C.

2. Wash 15 min in 2xSSCTw at 55C or at 65C.

3. Wash 2 x 30 min in 0.2xSSCTw at 55C or at 65C.

VI. Detection

All detection steps are performed at RT and on a shaker except the staining reactions.

1. Block for 1-8 h with PBSTw plus 5% sheep serum.

2. Incubate embryos in 100 ul preabsorbed sheep anti-Fluorescein-AP Fab fragments at a 1:2000 dilution in PBSTw. You can reuse antibody 2x.

3. Shake for 2 hr at RT or overnight at 4C. Save used antibody in a new tube designated 1x used or 2x used.

4. Wash 6 x 20 min with PBSTw (you can perform one of the washes overnight).

5. Wash 2 x 5 min in 0.1 M Tris-HCl pH 8.2, 0.1% Tween.

6. Dissolve Fast Red tablets in 0.1 M Tris-HCl pH 8.2, 0.1% Tween (2 ml/tablet) and sterile filter.

7. Stain in Fast Red solution for up to 48 h.

8. Wash 3 x 5 min with PBSTw. (Storage at 4C possible.)

9. Incubate 10 min in 0.1 M glycine-HCl pH 2.2 plus 0.1% Tween at RT to remove first antibody.

10. Wash 4 x 5 min in PBSTw.

11. Incubate embryos in 100 ul preabsorbed sheep anti-Digoxigenin-AP Fab fragments at a 1:2000 dilution in PBSTw. You can reuse antibody two times.

12. Shake for 2 hr at RT or overnight at 4C.

13. Wash 6 x 20 min with PBSTw (one of the washes overnight possible).

14. Wash 2 x 5 min in SB.

15. Stain in SS for up to 48 hr.

16. Wash 3 x with PBSTw.

17. Embryos may be fixed for at least 30 min and stored at 4C in PBSTw (add NaNO3).

18. Mount in glycerol.

Do not clear embryos in alcohol because Fast Red precipitate is purported to be unstable in ethanol.

Comments

To detect two different RNA transcripts in whole embryos make one RNA probe using Fluorescein-UTP and the other using Digoxigenin-UTP. Both probes are hybridized at the same time and detected one after another in two rounds of detection. The crucial step is the inactivation and removal of the first applied Fab-AP conjugate using the low pH step after the first staining round. Inactivation of the first applied Fab-AP conjugate by heating is not recommended, since incubation at 65C for 20 min or at 80C for 10 min causes fading of the red precipitate resulting in a weaker and more diffuse signal. In contrast, the blue precipitate darkens resulting in an increased background in the embryo and a nearly black yolk.

Because Fast Red is less sensitive than BCIP/NBT (5-10x) the stronger probe is always visualized in red. Because the second round of detection is less sensitive than the first, stain the red first. Nevertheless, if necessary, it is also possible to do the purple staining first. Fluorescein labeled probes are slightly less sensitive than digoxigenin probes. Therefore the stronger probe is usually detected in red using a fluorescein probe. To detect transcripts of a single gene use just one probe and one round of detection with BCIP/NBT as substrate.

Antibodies, Stock Solutions and Reagents

Preadsorption of antibody with zebrafish embryos

Zebrafish embryos need not to be dechorionated. The number of embryos you use for preabsorption depends on the amount of antibody you want to preabsorb. The stages used for preabsorption should include the same or older stages you will use in your in situ hybridizations. 2-3 d embryos work well.

1. Fix embryos in 4% paraformaldehyde at 4C overnight.

2. Wash 4 x 5 min in PBSTw.

3. Store embryos in 100% methanol at -20C.

4. Rehydrate 1 ml of embryos by rinsing 3 x 5 min in PBSTw.

5. Transfer embryos to a 2.0 ml Eppendorf tube.

6. Homogenize embryos with a pestle and adjust to about 1.0 ml with PBSTw.

7. Add 10 ul antibody (final 1:100).

8. Preabsorb antibody by shaking at least overnight or store for a few days at 4C. Spin down embryonic debris and sterile filter (Millex-GV 0.22 um, Millipore) supernatant.

9. Resuspend embryonic debris in PBSTw and sterile filter again.

10. Fill up with PBSTw to a final 1:2000 dilution of antibody.

11. Store preabsorbed antibody (20 ml) at 4C.

12. The antibody preabsorbed with zebrafish embryos works well also for staining of Drosophila embryos.

Stock solutions and other reagents
0.5 M PO4 buffer pH 7.3: 80 ml 0.5 M Na2HPO4 + 20 ml 0.5 M Na2HPO4
10x PBS: 8% NaCl, 0.2% KCl, 0.2 M PO4buffer pH 7.3
1xPBSTw: 1x PBS + 0.1% Tween-20
100 mg/ml glycine in ddH2O: store at -20C
20 mg/ml proteinase K in ddH20: store at -20C in aliquots
4% paraformaldehyde in 1x PBS is prepared under a fume hood:

  1. mix in a beaker: 2 g paraformaldehyde, 5 ml 10x PBS, 45 ml ddH20
  2. heat to 70C
  3. stir for about 2 hr until all paraformaldehyde is dissolved
  4. cool to 4C
  5. add 4 ul 1N NaOH
  6. mix and store at -20C in 5 ml aliquots
HB4: 50% formamide (Merck), 5x SSC, 50 ug/ml heparin, 0.1% Tween-20, 5 mg/ml torula RNA (Sigma)
50 mg/ml heparin: store at -20C
2x SSCTw: 2xSSC + 0.1% Tween-20
0.2x SSCTw: 0.2xSSC + 0.1% Tween-20
Sheep serum: heat inactivate at 56C for 30 min and store at -20C
Fast Red tablets (Boehringer): 0.5 mg napthol substrate, 2 mg Fast Red chromogen, 0.4 mg levamisole per tablet
SB: 100 mM NaCl, 50 mM MgCl2, 100 mM Tris-HCl pH 9.5, 1mM levamisol, 0.1% Tween-20
NBT (Boehringer): 75 mg/ml in 70% DMF/H2O
BCIP (Boehringer): 50 mg/ml in 100% DMF
SS: 4.5 ul NBT + 3.5 ul BCIP in 1 ml SB Final: 337.5 ug/ml NBT and 175 ug/ml BCIP

Reference: Hauptmann, G. and Gerster, T. (1994) 2 color whole-mount in situ hybridizations on zebrafish and Drosophila embryos. Trends Genet. 10: 266.


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