Gastrin-Releasing Peptide-Expressing Nerves Comprise Subsets of Human Cutaneous Aδ and C Fibers that May Sense Pruritus

obtained from Jackson Immunoresearch (West Grove, PA), were raised in donkey and conjugated with biotin, horse radish peroxidase, or Dylight dyes. Skin Specimens and Immunohistochemistry: Human skin, free from skin-disease, scar or other signs of trauma, and without incidental lesions, was obtained from the dog ears of excision specimens from both benign and malignant lesions of 17 patients, otherwise without pruritic skin diseases (following approval by the IRB at Boston University to use this de-identified and otherwise discarded surgical tissue without informed consent) and according to Declaration of Helsinki Principles). Patient consent for experiments was not required because Some of these skin samples were taken from patients with non-melanoma skin cancers, and these patients can have both pain and itch (Mills et al.). While no specific itch or pain rating was performed prospectively on the human skin from this study, the dog ear excision specimens were asymptomatic. Mouse skin was obtained from the cheek of 3 male C57/Bl6 mice (following approval by the IACUC at Boston University). Human and mouse skin were placed directly into PLP fixative for 24 hrs, then cryoprotected with 20% sucrose in 0.1M Sorrenson's buffer, frozen with dry ice, and then sectioned with either a sliding microtome or a cryostat. The immunohistochemical procedure to detect cutaneous nerves was similar to previous described methods (Kennedy and Wendelschafer-Crabb, 1993). Frozen sections 80 uM thick were processed for triple-labeled immunofluorescence localization as follows: No antigen retrieval was used. Sections were blocked for 2 hours in the 0. Horseradish peroxidase was inactivated with hydrogen peroxide incubation then the remaining primary antibodies were incubated as a cocktail, and either detected with a dylight–conjugated secondary