Alternative Methods for Fluorescent Detection

The main protocol presented on the previous pages combines two ways to detect RNA probes with fluorescence. However, it's possible to add some other techniques into the mix, as described below. For more comments about 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 you might want 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. Next, inactivate the peroxidase that has just been used: Rock 1x, (PBT+ 1% H2O2), 20 minutes.
  3. Then 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:

We completely circumvented the problems associated with species separation of antibodies by using 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 are incubated with the embryos. The experiment looked like this:

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

and the result can be seen in this embryo image. Follow the kit instructions to make the zenon complexes, then add the different complexes to the embryos at step 6 in Post-Hybridization Fluorescent Detection. Then go directly to the final washes at step 11, and you're done! It's very easy, you can use those three very good mouse monoclonals together, and the signal-to-noise ratio of the stain is excellent. The signal might not be quite as bright as you get with the traditional primary/secondary method, but it's close and it doesn't really matter since the 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 perhaps it would be best to keep the incubation time to 1 hour. I did 1.5 hours at room temperature with a 1:200 dilution of all three complexed antibodies, and saw absolutely no cross-talk between the expression patterns of the genes being detected. So the antibody complexes seem to be very tightly glued together, but there might be a problem doing the incubation overnight at 4° C., we'll see.

Direct fluorescent labeling of RNA probes:

Another method with great potential is using riboprobes that are themselves labeled with Alexa fluors. After the post-hybridization washes, including only 15 minutes of PBT washes, fluorescent probes are bound to their targets and the embryos are ready to look at. I've had moderate success using this direct-label method, but the signals are significantly weaker than I can get with the indirect label methods and for some reason I'm having background problems, too. But it definitely works, as shown in this embryo image, and can be combined with the other methods described here. 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), and I'll be putting up a protocol here.

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