Simultaneous Detection of mRNA and Protein
The challenge in trying to detect both protein and mRNA in your embryos is finding the conditions which will yield an acceptable result for each. The mild proteinase digestion of the embryos results in the optimum hybridization of probes to their targets, giving you the best RNA signal, but will destroy many of the protein epitopes that your antibody should recognize, giving you the worst protein signal, or none at all. Here are some methods that get around the ProtK treatment, thus allowing your antibody to work while still obtaining a good signal from your RNA probe.
- First, Nagaso et al. (2001) introduce the use of acetone as a ProtK replacement for simultaneous protein and mRNA detection in both embryos and wing discs. Briefly, the samples are treated with xylenes/ethanol and then with acetone, followed by a single post-fixation and pre-hybridization. Since a xylenes/ethanol treatment is already included in my normal in situ protocol, I also tried to simply replace the ProtK step with the acetone treatment as specified in this paper, as follows:
- At the ProtK step, instead Wash 2x, ddH2O
- Incubate in 80% acetone / 20% ddH2O, 10 minutes at -20° C.
- Wash 2x, PBT; Rock 1x, PBT, 5 minutes
- Proceed to the second post-fixation.
- The second idea I've heard about is a brief heat treatment in place of the ProtK digestion. The following steps are modifications of a protocol from Uwe Lammel (Klambt lab), and you can vary the time and temperature in order to optimize the antibody stain and embryo morphology.
- At the ProtK step, instead Wash 2x, 1x PBS (no Tween)
- Incubate in a water bath at 90°-95° C. for 2-3 minutes; stir the embryos once for a few seconds during this incubation. The volume
of embryos grows as they expand with heating.
- Incubate on ice for 5 minutes
- Wash 1x, PBT, and proceed to the second post-fixation.
- If you have blazing signal from your RNA probe, simply eliminate the ProtK digestion and the second post-fixation, skipping directly to the pre-hybridization after the first post-fixation and PBT washes. The RNA signal-to-noise ratio will suffer, but it will probably be OK. To compensate for reduced signal, you could try using tyramide signal amplification. This approach was used by Knirr et al. (1999) and Wu et al. (2001) to detect both transcripts and protein in embryos. Another approach is described in a protocol by T. Jowett (2001), in which ProtK is not used and the hybridization temperature is 70° C.; perhaps at this higher temperature the probes regain accessibility to the tissue normally provided by the ProtK treatment.
These methods assume that your antibody will still work well even after its protein targets have been slow-cooked in hybridization solution. For the antibodies I have tried, the hybridization itself does not seem to reduce the quality of antibody staining, but that might not always hold true. So what works best? All three methods have worked for me, but I haven't tried them in parallel so I can't make a fair comparison. They're all worth trying with your particular RNA probe and antibody combination. Parallel experiments with and without ProtK made me conclude that ProtK treatment is still essential for the best RNA signal and you will have to sacrifice some percentage of it in order to see your protein as well. If you have great signal to begin with, you will get your cake and eat it too. But what if the harsh conditions of hybridization do completely destroy the epitopes normally recognized by your antibody? Another approach is to perform a normal antibody staining procedure before hybridization. Subsequent post-fixation of the embryos allows the secondary detection reagents to persist through the hybridization steps, to be visualized later along with the RNA probe. Two variations on this theme are described in Goto and Hayashi (1997) and Sturtevant et al. (1993).
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