Simultaneous Detection of mRNA and Protein
The challenge in trying to detect both protein and mRNA in 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 riboprobes to their targets, resulting in the best RNA signal, but can destroy many of the protein epitopes recognized by antibodies. The following methods can substitute for the ProtK treatment, thus avoiding the destruction of epitopes while retaining a good signal from the RNA probe.
- Nagaso et al. (2001) use acetone in place of ProtK 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 the normal in situ protocol, one can simply replace the ProtK step with the acetone treatment, 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.
- A brief heat treatment can also be tried instead of the ProtK digestion. The following steps are modifications of a protocol from Uwe Lammel (Klambt lab). Time and temperature can also be varied 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 the signal from the RNA probe is very strong, one can try eliminating the ProtK digestion and the second post-fixation, proceeding directly to the pre-hybridization after the first post-fixation and PBT washes. The RNA signal-to-noise ratio will diminish somewhat, but probably will be acceptable. To compensate for reduced signal, tyramide signal amplification (TSA) can be used. 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.
- The preceeding methods require that the antibody still perform well even after its protein targets have been subjected to the long hybridization. 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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