A Novel Antiserum Against a Predicted Human Peripheral Choline Acetyltransferase (hpChAT) for Labeling Neuronal Structures in Human Colon

This study was carried out in accordance with the recommendations of “NIH Guide for Care and Use of Laboratory Animal protocols” and of the Institutional Animal Care and Use Committee of Shiga University of Medical Science (SUMS). The protocol was approved by SUMS. Six adult male Balb/c mice (5-weeks-old) were purchased from Clea Japan Inc. (Konan, Japan). Each mouse (labeled H1-H6) received subcutaneous injections of 0.1 ml of TiterMax Gold adjuvant (Sigma-Aldrich, Saint-Louis, MO, USA) containing 5 mg/ml KLH-conjugated peptide into multiple sites of the neck and back skin with at a 2-week interval. Antisera, collected 4 days after immunization or booster injections were tested by IHC on sections of human colon. After 9–13 booster injections, the whole blood was collected from the heart under deep anesthesia with an intraperitoneal injection of sodium pentobarbital (80 mg/kg, Somnopentyl, Kyoritsu Seiyaku, Tokyo, Japan). After exsanguination, animals were euthanized by intracardiac injection with a lethal dose of sodium pentobarbital (150 mg/kg). The antiserum was isolated from collected mouse blood using a blood-serum separation device (Nipro, Osaka, Japan). All mice immunized gave high-titer antisera capable of labeling positive structures in human colon by IHC (Supplementary Figure S1). Among them, the mouse H3 gave the highest titer antiserum after 13 booster injections. Then, this H3 antiserum, termed here hpChAT antiserum, has been used in the p...
Source: Frontiers in Neuroanatomy - Category: Neurology Source Type: research