Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.
1. FIXATION
NOTE REGARDING PFA:
We store 4% PFA in aliquots at –20ºC (see table of reagents). For the initial fixation, we only use PFA that has never been previously thawed. For subsequent fixations, we often use PFA that has been previously thawed. The initial fixation appears to be critical for successful staining with this protocol.
2. PROTEINASE AND POSTFIXATION
3. PREHYBRIDIZATION
4. HYBRIDIZATION
5. PROBE REMOVAL
Note that the solutions from this point forward lack detergent. Elimination of detergent appears to help the staining reactions but does cause the embryos to become rather sticky.
6. ANTI-FLUORESCEIN ANTIBODY INCUBATION
7. DETECTION OF FLUORESCEIN-LABELLED PROBE
NOTES ON THE TYRAMIDE SIGNAL AMPLIFICATION:
We have been using the Perkin Elmer TSA Kits. We find that the Alexa-Tyramide substrates stain well using the Perkin Elmer amplification diluent buffer, but we have not had success using the staining buffer provided with the Invitrogen/Molecular Probes Kits. Lastly, we have found that Cy5 fluorescence is eliminated by subsequent Methanol/H2O2 treatment while fluorescein and Alexa-647 are unaffected. Cy3 may also be adversely affected by the Methanol/H2O2 treatment as it is structurally related to Cy5. For this reason, Cy3 and Cy5 TSA reactions should only be used for the second staining reaction in a double fluorescent in situ protocol.
8. ANTI-Digoxygenin ANTIBODY INCUBATION
9. DETECTION OF Digoxygenin-LABELLED PROBE
10. PROPIDIUM IODIDE STAINING
11. MOUNTING
SOLUTIONS:
PBST | PBS plus 0.1% Tween | |
SSCT | SSC plus 0.1% Tween | |
HYB- | 50% formamide 5xSSC 0.1% Tween-20 |
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HYB+ | HYB- 5mg/ml torula (yeast) RNA 50μg/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. |
1 x maleic acid buffer | 150mM maleic acid, 100mM NaCl (pH 7.5) |
Figure 1. Representative results of whole mount double fluorescent in situ hybridization. (A-C). Images show a single confocal section through the posterior region of a zebrafish embryo at the ten-somite stage. The view is dorsal with anterior to the top. Nuclei stained with propidium iodide are colored blue. (A) deltaC mRNA was detected using a digoxygenin-labeled riboprobe and TSA-Cy5 reagent. (B) mRNA transcribed from the her1 gene was detected with a fluorescein-labeled riboprobe and TSA-fluorescein reagent. (C) Merging the green and red channels unambiguously identifies regions of distinct or overlapping gene expression of the two genes. (D, E) Detail of her1 expression demonstrating the high resolution of this procedure. Subcellular localization of mRNA can be clearly discerned. Arrowheads indicate a cell exhibiting active transcription, revealed by dots of staining in the nucleus. Arrows indicate a cell showing cytoplasmic localization of mRNA. (F, G) Double staining for her1 and deltaC reveals cells that are transcribing both genes (white arrowhead), or either gene separately (red and green arrowheads).
The protocol presented here works well with probes that give a clean strong signal after staining for 30-45 minutes in a typical alkaline phosphatase-mediated reaction. Prior to performing the fluorescent in situ hybridization protocol, we always test our probes using the standard non-fluorescent protocol (provided in supplemental material along with the probe synthesis protocol). We have had less success with the fluorescent in situ hybridization protocol when using weaker probes, but it may be possible in some cases. We have successfully combined the fluorescent in situ hybridization protocol with immunolocalization of β-catenin 3. When performing this variation, we do the HYB-, HYB+ and hybridization incubations at 55°C instead of 65°C as the epitopes appear to be preserved better at the lower temperature. The immunolocalization incubations are performed after the TSA reactions.
Since fluorescein-labeled probes are generally weaker than a digoxygenin-labeled probe for the same gene, we usually visualize the fluorescein-labeled probe first. One instance where one might want to visualize the digoxygenin probe first is if one of the two genes is expressed at a low level. In this case, one makes a digoxygenin-riboprobe for the weak gene and stains for it first to maximize the signal to noise. Note that if you visualize the fluorescein probe second, you should not visualize the digoxygenin probe with fluorescein TSA as the anti-fluorescein antibody will recognize the TSA stain as well as the fluorescein-labeled riboprobe.
We would like to acknowledge the contributions of Dörthe Jülich, Jennifer Round and Andrew Mara in developing this protocol. Research support provided by the NICHD, the American Cancer Society and the March of Dimes.
Material Name | Type | Company | Catalogue Number | Comment |
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Paraformaldehyde 16% solution, EM Grade | Electron Microscopy Services | 15710 | Dilute to 4% in 1.33x PBS (final 1x). Store in 2ml aliquots at –20°C | |
Proteinase K, recombinant PCR grade | Roche | 03115879001 | Make a 20mg/ml stock solution in water. Store in 1ml aliquots at –20°C | |
Blocking Reagent | Roche | 11096176001 | Make a 10% stock solution in 1x maleic acid buffer. Store in 50ml aliquots at –20°C. Stable over multiple freeze/thaws. | |
Anti-Fluorescein-POD, Fab fragments | Roche | 11426346910 | Aliquot and store at -20°C. Keep one working aliquot at 4°C. | |
Anti-Digoxigenin-POD, Fab fragments | Roche | 11207739910 | As above. | |
TSA Plus Fluorescein system | Perkin Elmer | NEL741001KT | Prepare each vial of TSA reagent only when needed. Dissolve in 60μl DMSO. Store at 4°C. | |
TSA Plus Cyanine5 system | Perkin Elmer | NEL745001KT | As above | |
TSA Plus Cyanine3 system | Perkin Elmer | NEL744001KT | As above | |
TSA Kit #16 AlexaFluor647 tyramide (plus HRP-goat-anti-rabbit IgG) | Molecular Probes /Invitrogen | T20926 | Dissolve tyramide reagent in 150μl DMSO, aliquot and store at –20°C. | |
RNase, DNase free | Roche | 11119915001 | Supplied as 500μg/ml solution | |
Propidium Iodide | Molecular Probes /Invitrogen | P-3566 | Supplied as 1mg/ml solution in water. |