Simultaneous detection of receptor mRNA and ligand protein in human skin tissues

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Abstract
Background: In situ hybridization techniques allow a cell-type-specific messenger RNA (mRNA) analysis in complex tissues such as human skin. Methods: To evaluate both the expression of mRNA and protein within the same tissue section, we developed a protocol of combined non-radioactive in situ hybridization and immunohistochemistry for use in dermatohistopathology. To validate the technique, we assessed the distribution of vasoactive intestinal polypeptide (VIP) protein and its receptor, VPAC2 mRNA, in human skin samples. Results: Simultaneous detection of VPAC2 mRNA and VIP immunoreactivity led to abundant staining for both signals in a variety of cell types. There was marked staining for VPAC2 mRNA in epidermal cells, with most pronounced hybridization signals found in keratinocytes of the basal layer and in glandular cells surrounded by VIP-immunoreactive nerve fibers. Hair follicle cells next to VIP-positive fibers also exhibited hybridization signals. Specific staining was also detected in endothelial and mononuclear cells. The findings of simultaneous in situ hybridization and immunohistochemistry were identical to results obtained by the single application of both methods. Conclusions: The combination of in situ hybridization and immunohistochemistry appears to be a promising technique to assess the expression of both protein and mRNA in skin samples and may be used for various purposes in experimental and clinical dermatopathology.