Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: V. A methodology for examining correlative monoamine-neuropeptide neuroanatomy

Abstract

A technique for the simultaneous visualization of monoamines and neuropeptides is described. This technique provides for the correlative examination of monoamine and neuropeptide interrelationships either simultaneously using two adjacent sections of freeze-dried tissue or sequentially using a single freeze-dried tissue section for both histofluorescence and immunocytochemistry. Both techniques provide for the investigation of precise anatomical interrelationships and used together provide confirmation and complementary information concerning neuropeptide-monoaraine interactions. Histological comparisons between freeze-dried and Bouin's fixed tissue revealed three differences: First, the cytoplasm of neurons in freeze-dried tissue retained a reticulated appearance; secondly, neurons in freeze-dried tissue demonstrated a greater affinity for chemical stains; and thirdly, tissue fixed in Bouin's solution appeared shrunken when compared to tissue which had been freeze-dried. Application of immunocytochemistry to freeze-dried tissue proved successful with all antisera tested and the quality of the immunostaining was similar in both freeze-dried and Bouin's fixed tissue. These observations suggest that freeze-drying fixation provides a suitable tissue preparation for immunocytochemistry and a technical means for coupling immunocytochemical staining of neuropeptides and fluorescence histochemical methods for monoamines in a single tissue block.