Histological Localization of Alcohol Dehydrogenase: Methods, Approaches and Applications

© 2004 Elsevier Ltd. All rights reserved. This chapter discusses the methods for histological localization of alcohol dehydrogenase. Tissue localization of multiple enzyme forms of human Alcohol Dehydrogenase (ADH) is of paramount importance to understand their physiological involvement in ethanol metabolism and organ susceptibility to alcohol damage. Histological techniques-such as immunohistochemistry and in situ hybridization, provide powerful and complementary approaches to identify definite cell layers or cell types that may contribute to ethanol elimination and, for that matter, be subjected to the local toxic effects of ethanol. Immunohistochemical methods rely on the availability of specific antibodies against different ADH forms. When polyclonal antiserum is the starting material, antibodies specific for a given ADH should be affinity purified and thoroughly checked for cross-reactivity against other ADH forms and proteins by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In situ hybridization methods, being highly sensitive, should use specific homologous probes, which have been previously characterized by Northern blot analysis. With either technique, it is essential to perform adequate control experiments by using pre-immune serum, pre-absorbed antiserum or sense RNA probes. Experimental conditions should always be carefully optimized and it is advisable that results be validated with appropriate functional tests or activity assays.