ABSTRACT
In situ hybridization histochemistry (ISHH) is a flexible molecular biologic technique which can be used to identify the distribution of receptors in organs and tissues. ISHH localizes to the cell body and identifies cells synthesizing the receptors, which is an advantage over conventional radioligand autoradiography and immuno-histochemistry. This chapter utilises the technique of ISHH to study rapid-eye-movement sleep deprivation effects on m1–m3 muscarinic receptor messenger ribonucleic acids (mRNA) expression in the rat brain. Muscarinic receptors are classified by their affinity for different cholinergic antagonists. Following removal, blocking, and freezing, the brain is sectioned in a cryostat and the sections are placed directly on the prepared slides. With fixed tissue, the mRNA may be unavailable to the probe; thus, tissue deproteination by proteinase K increases probe access. The tissue is placed in a buffer solution, acetylated to block the positive charges from the lysine, rinsed, and then dehydrated with ethanol.
