Localization of Y-Receptor Subtype mRNAs in Rat Brain by Digoxigenin Labeled In Situ Hybridization

In situ hybridization histochemistry (ISHH), first described in 1969 (1 ,2 ), allows a specific complementary RNA species to be detected directly at its site of expression, revealing its cellular localization and relative abundance. The utilization as a label of digoxigenin (3 ), which is not endogenous to mammalian tissue, provides a sensible alternative to radiolabels, having obvious inherent advantages (e.g., safety, speed, and higher cellular resolution), yet provides comparable sensitivity (4 –7 ). The method basically includes the following six steps: (1) probe labeling (the cloning techniques needed to produce suitable vector templates for cDNA and riboprobe synthesis are not covered in this chapter); (2) tissue preparation; (3) prehybridization tissue treatment; (4) hybridization; (5) posthybridization washing; and (6) signal detection.