Correlative histochemical and immunocytochemical techniques for the study of classical transmitters and neuropeptides

Abstract

A growing number of small biologically active peptides has been discovered in the nervous system and the endocrine organs of different mammalian species. These so-called neuro- or regulatory peptides may function as neurotransmitters, hormones or trophic factors. In many cases neuropeptides are colocalized in the same neurons and endocrine cells with classical transmitters and hormones. To understand the functions of neuropeptides it is important to determine the exact localization of neuropeptides in tissues and to clarify their relations to classical transmitters. In this paper a method for correlative visualization of epinephrine and norepinephrine with formaldehyde-glutaraldehyde (Faglu) fluorescence and subsequent demonstration of neuropeptides at light and electron microscopic level with pre-embedding immunocytochemistry in the same Vibratome section is described. A method for correlative demonstration of acetylcholinesterase (AChE) and neuropeptides at light microscopic level is also presented.Adrenal glands were fixed with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1M phosphate buffer, pH = 7.3. The adrenals of small rodents such as rat and hamster were fixed by immersion fixation for 6 h. The adrenals of larger species like cats and guinea pigs were fixed by perfusion for 20 min and further fixed by immersion for 1 hr. After fixation the tissues were rinsed several times in phosphate buffer and 7- to 10-um-thick sections were cut with a Vibratome. Sections are embedded in 0.5M Tris-saline buffer, pH = 7.6, which contained 20% glycerol and the catecholamine fluorescence was photographed (Fig. 1). After photography the coverslip was removed and the sections were processed free-floating through the steps of the pre-embedding modification of the PAP-method. After immunocytochemical staining the sections were post-fixed with 2% aqueous osmiumtetroxide for 1 h and dehydrated in graded ethanol series and flat-embedded in Epon under plastic coverslip. The areas photographed for catecholamine fluorescence were identified and photographed (Fig. 2). Desired areas were processed for electron microscopic examination. Human sympathetic ganglia were obtained from surgery and fixed with 4% paraformaldehyde for 6 h. Tissues were cryoprotected with 10% sucrose and 5 um frozen sections were cut on glass slides. Immunocytochemistry was carried out with indirect immunofluorescence method. After immunocytochemistry the sections were reacted for AChE according to the method of Karnovsky and Roots. Rhodamine-fluorescence and AChE were photographed using appropriate filters.