Immunohistochemical localization of polypeptides in peripheral autonomic nerves using whole mount preparations

Abstract

A method is described for the immunohistochemical localization of peptides in whole-mount preparations. Tissue is fixed as laminae with a picric acid/formaldehyde mixture and then dehydrated, cleared and rehydrated before exposure to antibodies. This procedure ensures adequate penetration of the antibody molecules without the need to freeze and thaw the tissue or to use detergents, preserves antigenicity and lowers non-specific background staining. The laminae are incubated with the primary antisera for 16 h at room temperature and, after washing, with a second, fluorescent tagged, antiserum. This can be followed by a peroxidase-anti-peroxidase localization of the second antiserum, which acts as a bridge. The method gives a precise and reproducible localization of immunoreactive peptides, with good penetration and low background even in thick preparation. Large areas can be scanned and neuroeffector relationships studied more easily than in sections.