A Nonradioactive Method of In Situ Hybridization That Uses Riboprobes and Paraffin-embedded Tissue and Its Combination with Immunohistochemistry

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Abstract
Current research into cytokine production in tissue sections relies on the detection of cytokine proteins using a variety of immunohistochemical methods. The disadvantages of this technique are that precise localization to a particular cell is difficult and it is uncertain whether the cells detected by this method are the origin or target of the cytokine or rather have nonspecifically absorbed the secreted cytokine. This question can be clarified using in situ hybridization, but current techniques are insensitive, poorly localizing, or time consuming. Biotin-labeled riboprobes were generated from cDNA fragments sandwiched between two RNA polymerase promoters (SP6 and T7 RNA polymerases) using a commercial riboprobe generation kit containing biotin-labeled UTP. The in situ hybridization technique was used to demonstrate cytokine mRNA in a range of tissues containing an inflammatory infiltrate and with a range of cytokine probes. This technique of in situ hybridization was combined with immunohistochemistry using an immunoalkaline phosphatase technique to show the powerful combination of these two techniques. The biotin-labeled riboprobes were sensitive enough to detect a range of cytokine mRNAs in a variety of tissue sections. The technique can be completed over a 24-h period and produces a stable color product that can be stored for long periods and can be quantitated using image analysis techniques. This technique was performed on paraffin-embedded tissue as well as cryosections and allowed for the detection of mRNA in archival tissue. It was also successfully combined with immunohistochemical techniques to determine simultaneously the localization of a cytokine product in particular cell lineages. A nonradioactive method for in situ hybridization using biotin-labeled riboprobes is described; it is capable of detecting mRNA products from a range of genes in a variety of tissue samples. An amplification step in the method enhances the sensitivity to a level that approaches that of radioactive methods, while maintaining the speed, safety, and simplicity of an immunoperoxidase detection system. The ability to use paraffin-embedded tissue with this method allows for improved tissue architecture and examination of archival tissue. These features should ensure greater use of in situ hybridization techniques in future research studies.

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