Immunocytochemical staining of neuropeptides in terminal arborization of primary afferent fibers anterogradely labeled and identified at light and electron microscopic levels

Abstract

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 1–11 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 μm 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 μm and the labeled arbors matched with those previously drawn from the 50-μm sections. Ultrathin sections were cut from the 5-μm 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.