IMMUNOCYTOCHEMICAL STAINING OF NEUROPEPTIDES IN TERMINAL ARBORIZATION OF PRIMARY AFFERENT-FIBERS ANTEROGRADELY LABELED AND IDENTIFIED AT LIGHT AND ELECTRON-MICROSCOPIC LEVELS

• Type: Article in International Scientific Journal
• Date: 1992

Authors

MERIGHI, A (Author) Other; Francisco Cruz (Author) FMUP; COIMBRA, A (Author) Other

Journal

Title: Journal of Neuroscience Methods

Vol. 42

Pages: 105-113

ISSN: 0165-0270

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 19 Citations

Scopus - 18 Citations

Other information

Authenticus ID: P-001-PQ8

DOI: 10.1016/0165-0270(92)90140-9

Abstract (EN): 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-II(o) 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-mu-m 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-mu-m and the labeled arbors matched with those previously drawn from the 50-mu-m sections. Ultrathin sections were cut from the 5-mu-m 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 9