Alternative Methods for Fluorescent Detection

The core protocol presented on the previous pages describes two methods for detecting multiple RNA probes with fluorescence: secondary immunofluorescence and tyramide signal amplification. However, additional fluorescent labeling methods can be used in this protocol, as described below. For more discussion of the relative merits of all the techniques presented here, see Conclusions and Future Problems.

Sequential fluorescent tyramide reactions

Since tyramide signal amplification (TSA) is a very sensitive method for fluorescent detection, in some cases it may be desirable to detect more than one target in this way. After performing the first TSA reaction on a sample, the activity of the peroxidase is eliminated, and then the sample is labeled again with a second fluorescent tyramide of a different color. As shown on the Protocol Overview, the first TSA reaction can be performed on the first day after hybridization, and the second on the following day:
  1. At step 6 in Post-Hybridization Fluorescent Detection, incubate the embryos with the first horseradish peroxidase conjugate at room temperature, and then perform the first TSA reaction as described.
  2. Inactivate the peroxidase that has just been used: Rock 1x, (PBT+ 1% H2O2), 20 minutes.
  3. Remove the H2O2: Wash 3x, PBT; Rock 1x, PBT, 5 minutes.
  4. Return to step 6 referred to above, and incubate with another HRP-conjugated detection reagent (along with anti-hapten antibodies, if necessary) overnight at 4° C., and finish the procedure as described, performing a second TSA reaction the next day with a different fluorescent tyramide.
The quenching of HRP with H2O2 between TSA reactions is described in the protocols for mouse tissue sections by Paratore et al. (1999) and Zaidi et al. (2000). Alternatively, a sequential TSA method developed for Drosophila embryos by Wilkie and Davis (1998) uses a 10 minute incubation in 0.01 M HCl to inactivate the first HRP.

Antibody labeling with Zenon complexes

A method which completely circumvents the problems associated with species separation of antibodies is based on the recently introduced 'Zenon' labeling reagents from Molecular Probes. Primary antibodies are incubated with fluorescent anti-IgG Fab fragments, and then these labeled antibody complexes, which remain tightly associated, are incubated with the embryos. An example of such an experiment is:

DIG probe --- Mouse anti-DIG-Alexa 647 zenon complex
BIO probe --- Mouse anti-BIO-Alexa 555 zenon complex
FITC probe --- Mouse anti-FITC-Alexa 488 zenon complex

(the result can be seen in this embryo image)

Follow the Molecular Probes kit instructions to make the zenon complexes, then add the different complexes to the embryos at step 6 in Post-Hybridization Fluorescent Detection. Then proceed directly to the final washes at step 11, and the staining is complete. This is a very straightforward method which makes it possible to use multiple mouse monoclonals simultaneously, and typically the signal-to-noise ratio of these stains is excellent. The signal is not as bright as with the traditional primary/secondary method, but it is more than acceptable and the fluorescent background is very low. One word of caution about the stability of the complexes: the protocol from Molecular Probes states that there may be some dissociation and re-association of complexes during the incubation period with the specimen, so it is best to keep the incubation time to 1 hour. For the experiment referred to above, an incubation time of 1.5 hours, at room temperature with a 1:200 dilution of all three complexed antibodies, resulted in no observable cross-talk between the expression patterns of the genes being detected.

Direct fluorescent labeling of RNA probes

Another potentially very powerful method is to use riboprobes that are directly labeled with Alexa fluors. After the post-hybridization washes, including only 15 minutes of PBT washes, fluorescent probes bound to their targets in embryos are ready to be detected. The signals obtained with directly labeled probes are significantly weaker than with indirect, amplified fluorescent labeling methods, and background staining is somewhat elevated. But for some genes a quite respectable signal-to-noise ratio is achieved (see this example embryo image). This method will be most useful for genes expressed at high levels, and can be combined with the other described labeling methods. Similar results with fluorochrome-labeled probes were reported in the protocol by T. Jowett (2001), in which a fluorescein-labeled RNA probe is directly visualized.

Making RNA probes incorporating fluorescent dye molecules is a two-step process. First, aminoallyl-UTP is used as the modified nucleotide in the normal transcription reaction. The RNA is purified, and then an Alexa fluor succinimidyl ester brings in the fluorescent dye molecule that is covalently linked to the RNA. After this modification, the RNA is purified again to remove the unbound dye ester, then hydrolyzed, precipitated, and hybridized to embryos as described. This method for making fluorescent probes stands in contrast to incorporating nucleotides modified with a fluorochrome into the RNA during the synthesis reaction, and results in a higher degree of labeling. For details on this protocol, refer to the Molecular Probes Handbook, sections 1.2, 8.2, and 8.5 ('ARES' nucleic acid labeling)

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