Immunocytochemical staining of neuropeptides in terminal arborization of primary afferent fibers anterogradely labeled and identified at light and electron microscopic levels.
A method is described to combine, at the ultrastructural level, horseradish peroxidase (HRP) anterograde tracing of primary afferents and peptide immunocytochemistry, using the lateral plexus of primary afferent fibers in laminae I-IIo of the rat dorsal horn as a model system. Free HRP was crushed against the dorsal roots. After a 14-h survival, animals were perfused, and the spinal cord was sliced at 50 microns with a Vibratome in a parasagittal plane. From these thick sections, camera lucida drawings of HRP-labeled fibers were obtained. Following osmication and Epon flat embedding, thick sections were re-cut at 5 microns and the labeled arbors matched with those previously drawn from the 50-microns sections. Ultrathin sections were cut from the 5-microns semithin sections and directly stained on grids using a post-embedding immunogold labeling procedure. Single and/or double immunocytochemical staining was performed using a rat monoclonal antibody against substance P and a rabbit polyclonal antiserum against calcitonin gene-related peptide (CGRP). Immunocytochemical reactions were visualized using appropriate immunoglobulin G-gold conjugates and the double-labeled synaptic boutons were matched with the varicosities previously visualized at the light level in the thick and semi-thin sections. The major advantages of this method are: (i) correlative studies at light and electron microscope level are made possible; (ii) tissue ultrastructure and antigenicity are adequately preserved so that a reliable subcellular localization of antigens under study is obtained; (iii) the markers used for tracing and immunocytochemistry are clearly distinguishable, even when present in the same nerve profile; and (iv) anterograde tracing can easily be combined with multiple immunolabeling.
Merighi
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Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Italy.
Cruz
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Coimbra
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D000818:Animals; D006735:Horseradish Peroxidase; D007150:Immunohistochemistry; D008297:Male; D008854:Microscopy, Electron; D009411:Nerve Endings / Q000648:ultrastructure*; D009475:Neurons, Afferent / Q000648:ultrastructure*; D009479:Neuropeptides / Q000737:chemistry* / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling; D013376:Substantia Gelatinosa / Q000648:ultrastructure*; D016610:Tissue Embedding
D009479:Neuropeptides; D006735:Horseradish Peroxidase