• Hybridization Histochemistry

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Hybridization histochemistry should enable the researcher to determine whether a given gene is expressed in particular cells. Figure 1 shows the simultaneous detection of two different transcripts detected through the use of radiolabeled and digoxigenin-labeled probes. Riboprobes are more sensitive than oligodeoxynucleotide probes, enabling one to see 5 or fewer transcripts per cell, and traditionally, ³⁵S-labeled probes are more sensitive than colorimetrically detected ones. However, as we gain more experience with amplification techniques, this superiority of radiolabeled probes may vanish. Furthermore, the recent introduction of the tyramide amplification system (TSA, also known as catalyzed reporter deposition or CARD) (Bobrow et al., 1989) offers a number of branch points for varying degrees of amplification and different reaction products (Kerstens et al., 1995; Hunyady et al., 1996).
X-ray or tritium sensitive films may provide the easiest means to quantitation if the signal is sufficient and the cells are closely grouped. In these cases, the film optical densities can be converted to copies of probe hybridized through the use of simultaneously exposed brain paste standards that incorporate known amounts of the radioisotope. Phosphorimaging devices (e.g., those of Fuji Medical Systems or Molecular Dynamics) offer 2 advantages over films: their sensitivity is up to 40-fold greater and the signals are directly proportional to the amount of hybridized radiolabeled probe. We generally examine our sections with the phosphorimaging system prior to dipping them into nuclear emulsion. Detailed protocols for quantitative analysis of autoradiograms are available (Gerfen, 1989; Young, 1992).
Hybridization histochemistry may be viewed as composed of three steps: preparation of the tissue sections, hybridization and washing of the sections, and detection of the hybridization signal. Preparation of the tissue sections, after collection of the tissue specimens, essentially consists of cutting the sections and, of course, depends upon the numbers of sections needed and the size of the region(s) studied. This may take hours to weeks.
The hybridization and washing steps take either 2 or 3 consecutive days, depending on whether radiolabeled or digoxigenin-labeled probes are used, respectively. Detection of the digoxigenin-labeled probes is then complete at the end of the third day. Depending on the signal strength and degree of resolution needed, radiolabeled probe deposition can be determined over the course of minutes using film or phosphorimaging plates to months after coating with nuclear emulsion.

Critical Parameters and Troubleshooting

Anticipated Results

Quantitative Analysis of Autoradiograms

Time Considerations

Literature Cited

1. Albertson, D.G., Fishpool, R.M. and Birchall, P.S. 1995. Fluorescence in situ hybridization for the detection of DNA and RNA.Methods Cell Biol 48:339-364.
   
2. Bobrow, M.N., Harris, T.D., Shaughnessy, K.J. and Litt, G.J. 1989. Catalyzed reporter deposition, a novel method of signal amplification.J Immun. Meth. 125:279-285.
   
3. Bradley, D.J., Towle, H.C. and Young, W.S. III. 1992. Spatial and temporal expression of alpha and beta thyroid hormone receptor mRNAs, including the b₂ subtype, in the developing mammalian nervous system.J. Neurosci. 12:2288-2302.
   
4. Burgunder, J.-M. and Young, W.S. III. 1988. The distribution of thalamic projection neurons containing cholecystokinin messenger RNA, using in situ hybridization histochemistry and retrograde labeling.Mol. Brain Res. 4:179-189.
   
5. Gerfen, C.R. 1989. Quantification of in situ hybridization histochemistry for analysis of brain function. In Methods in Neuroscience (Conn, P.M., ed) pp. 79-97. Academic Press, New York
   
6. Hunyady, B., Krempels, K., Harta, G. and Mezey, É . 1996. Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostainings.J. Histochem. Cytochem.12:1353-1362
   
7. Kerstens, H.M.J., Poddihe, P.J. and Hanselaar, A.G.J.M. 1995. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine.J. Histochem. Cytochem. 43:347-352.
   
8. Kiyama, H. and Emson, P.C. 1991 An in situ hybridization histochemistry method for the use of alkaline phosphatase-labeled oligonucleotide probes in small intestine.
   

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