摘要：

This chapter describes the procedures used to measure somatostatin mRNA by in situ hybridization. The chapter presents a detailed description of a methodology that provides rapid and reproducible counts of silver grains over individual cells to estimate cellular mRNA levels. In situ hybridization can be performed on either fresh-frozen or perfused tissue. The choice of tissue fixation depends on the type of tissue to be used and whether another procedure is to be performed in conjunction with the in situ hybridization. To obtain fresh tissue, the animal is asphyxiated with carbon dioxide and then decapitated. Prior to in situ hybridization, the brains are allowed to equilibrate in the cryostat chamber and then are trimmed and embedded in Tissue–Tck OCT. In situ hybridization histochemistry is performed with an RNA probe complementary to preprosomatostatin. The exposure time depends on the specific activity of the probe, the presumed abundance of the message to be detected and desired grain density. It is essential that control experiments be performed to verify the specificity of an in situ hybridization assay. An advantage of using in situ hybridization as opposed to other RNA detection methods is the preservation of anatomical specificity.

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