A pre-embedding triple-label electron microscopic immunohistochemical method as applied to the study of multiple...

For many neural regions it is of interest to know the identity of the target structures of two different types of inputs to that neural region. Such studies require use of a triple-label immunohistochemical method to differentially label the class of target structure and the two types of input so that they can be visualized at the electron microscopic (EM) level. We describe here a procedure for combining three different markers (diaminobenzidine, benzidine dihydrochloride, and silver-intensified immunogold) for triple-label EM immunohistochemical pre-embedding labeling. All three markers are distinct at the LM and EM levels. An example of this approach as applied to studying striatal input to the ventral tegmental area is presented and the advantages of this approach are discussed.