An Immunohistochemistry Protocol to Identify Substance P Neurons

Substance P (SP), calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4) are markers for peripheral sensory neurons with unmyelinated axons. It is important to understand plasticity in peripheral pain pathways, particularly in neonates when noxious stimuli are processed by pathways that are still maturing. Our laboratory [1] has shown that, in rats experiencing painful stimuli as neonates changes in nociception-related cortical motor, sensory and limbic areas persist for up to 180 day. This study aimed to validate an immunohistochemistry (IHC) method for dorsal root ganglion (DRG) neurons to further investigate the effects of neonatal pain on these cells. Six male Wistar rats aged 15 and 180 days were anesthetized and perfused with 4% buffered paraformaldehyde. L6 DRG were removed and frozen. Cryosections (16 µm) were incubated in rabbit anti-substance P (1:1.000; 24hs), biotinylated anti-rabbit IgG (1:500; 24hs) and finally Extravidin-HRP (1:1.500; 24hs). A nickel-intensified diaminobenzidine reac...