A modified peroxidase--antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord.

Abstract

A procedure is presented which modifies the Sternberger peroxidase--antiperoxidase (PAP) technique in order to visualize additional amounts of immunodeposits representing the antigen substance (SP) in 5-micrometer paraffin tissue sections of rat spinal cord. For increased sensitivity, the new procedure utilizes a "double bridge" and diaminobenzidine in low pH buffer. The modifications have made possible the visualization of immunoreactive beaded processes and punctate bodies, which were then traced to determine patterns of SP circuitry. Using the modified PAP procedure, the greatest number of immunoreactive processes appeared in the dorsal horn, where some punctate bodies and varicose processes could be seen adjacent to the myelinated afferent fiber bundles that penetrate the substantia gelatinosa as dorsal root collaterals. Additional immunoreactive processes and punctate bodies coursed through the myelinated afferent fiber bundles that penetrate the dorsolateral white matter, and extend into the intermediolateral gray region. Substance P was also identified within immunoreactive processes found in Rexed's laminae V and VI, as well as the central canal region, the dorsal gray commissure, and the ventral gray and white commissures. Since the modifications improved the visualization of SP-containing processes in sparsely populated regions of the spinal cord, especially the ventral horn, they may be useful in demonstrating other antigens that normally occur in small quantities within tissues.

Substance P and 5-hydroxytryptamine immunoreactive presynaptic boutons on presumed .ALPHA.-motoneurons in the chicken ventral horn.

“…Light microscopically, the present observations using monoclonal antibodies to the 5-HT and SP in the chicken ventral horn were consistent with previous observations in the ventral horn of various mammals and the human using conventional polyclonal antibodies to the 5-HT K0JIMA et al, 1982K0JIMA et al, , 1983 and SP (HOKFELT et al, 1975;LJUNGDAHL et al, 1978;VAccA et al, 1978VAccA et al, , 1980LAMOTTE and DE LANEROLLE, 1981). However, the size, shape and number per unit area of Immunoreactive structures on presumed ~-motoneurons were different for the SP and 5-HT containing boutons.…”

Stimulation of somatostatin expression in developing ciliary ganglion neurons by cells of the choroid layer

“…This is consistent with the notion that a target interaction controls somatostatin expression. Our use of the sensitive double PAP technique for immunocytochemical detection (Vacca et al, 1980;Ordronneau et al, 198 l), together with the ease of recognizing the discrete pun&ate intracellular localization of SOM-IR, made us confident that a lack of detectable SOM-IR within a neuron reflected an insignificant level of somatostatin expression.…”

Localization of androgen receptors and estrogen receptors in the same cells of the songbird brain.

“…As a modification to this protocol, a double-bridge technique was used to enhance the staining (13). Briefly, sections were treated with 0.1% Triton X-100 in 25 mM phosphate buffer containing 0.9% NaCl (PBS) for 30 min.…”