title: Verification of the Cross Immunoreactivity of A60, a Mouse Monoclonal Antibody against Neuronal Nuclear Protein.
abstract: A60, the mouse monoclonal antibody against the neuronal nuclear protein (NeuN), is the most widely used neuronal marker in neuroscience research and neuropathological assays. Previous studies identified fragments of A60-immunoprecipitated protein as Synapsin I (Syn I), suggesting the antibody will demonstrate cross immunoreactivity. However, the likelihood of cross reactivity has never been verified by immunohistochemical techniques. Using our established tissue processing and immunofluorescent staining protocols, we found that A60 consistently labeled mossy fiber terminals in hippocampal area CA3. These A60-positive mossy fiber terminals could also be labeled by Syn I antibody. After treating brain slices with saponin in order to better preserve various membrane and/or vesicular proteins for immunostaining, we observed that A60 could also label additional synapses in various brain areas. Therefore, we used A60 together with a rabbit monoclonal NeuN antibody to confirm the existence of this cross reactivity. We showed that the putative band positive for A60 and Syn I could not be detected by the rabbit anti-NeuN in Western blotting. As efficient as Millipore A60 to recognize neuronal nuclei, the rabbit NeuN antibody demonstrated no labeling of synaptic structures in immunofluorescent staining. The present study successfully verified the cross reactivity present in immunohistochemistry, cautioning that A60 may not be the ideal biomarker to verify neuronal identity due to its cross immunoreactivity. In contrast, the rabbit monoclonal NeuN antibody used in this study may be a better candidate to substitute for A60.
authors.affiliation: Department of Neurology, Renmin Hospital, Wuhan University Wuhan, China.
authors.forename: Shanping
authors.initials: S
authors.lastname: Mao
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Children's Hospital of Philadelphia, University of Pennslyvania Philadelphia, PA, USA.
authors.forename: Guoxiang
authors.initials: G
authors.lastname: Xiong
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Children's Hospital of Philadelphia, University of Pennslyvania Philadelphia, PA, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Neurology, Renmin Hospital, Wuhan University Wuhan, China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Dong
authors.affiliation: Department of Neurology, Renmin Hospital, Wuhan University Wuhan, China.
authors.forename: Baohui
authors.initials: B
authors.lastname: Liu
authors.affiliation: Philadelphia Veterans Affairs Medical Center, University of PennslyvaniaPhiladelphia, PA, USA; Departments of Otorhinolaryngology-Head and Neck Surgery, University of PennslyvaniaPhiladelphia, PA, USA.
authors.forename: Noam A
authors.initials: NA
authors.lastname: Cohen
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, Children's Hospital of Philadelphia, University of PennslyvaniaPhiladelphia, PA, USA; Department of Anesthesiology and Critical Care Medicine, Perelman School of Medicine, University of PennslyvaniaPhiladelphia, PA, USA.
authors.forename: Akiva S
authors.initials: AS
authors.lastname: Cohen
keywords: brain sections; epitopes; hybridoma; immunohistology; neurocytoma; stem cell differentiation