Fixation and demineralization of bone tissue for immunohistochemical staining of neuropeptides

The present study in the rat demonstrates the feasibility of applying immunohistochemical staining techniques on bone tissue for studies of substances such as neuropeptides contained in nerve fibers. Two fixation procedures, as well as the influence of demineralization on neuropeptide antigenicity, were studied in bone and for comparison in small intestine. In vivo perfusion with paraformaldehyde and picric acid, followed by demineralization in a solution of either EDTA-cacodylate or buffered EDTA-sucrose, proved to be the most appropriate with respect to preserved antigenicity. Antibodies to 23 neuronally related substances were tested. In the bone tissue, immunoreactivity was found to four neuropeptides: substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y, and also to the catecholamine-synthesizing enzyme tyrosine hydroxylase. The described method for identifying intraosseal neuropeptides offers a new means of studying skeletal innervation and bioactive substances in bone tissue.