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.

Seasonal changes in neuronal turnover in a forebrain nucleus in adult songbirds

“…Intestinal tissue was the control tissue of choice given constantly high numbers of mitotic cells present. With the NeuN antibody, we observed strong labeling in nuclei with occasional weak labeling in the cytoplasm of mature neuronal cells (Mao et al, ; Mullen et al, ; Saito et al, ). The rabbit‐anti‐NeuN antibody we use (Table ) was previously validated and shown to not cross‐react with A60 or Synapsin I (Mao et al, ).…”

“…Importantly, the MOBu‐1 BrdU antibody does not cross‐react with EdU labeled DNA (Liboska et al, ). NeuN is a neuron‐specific protein expressed in the soma of fully differentiated neurons of the central and peripheral nervous systems (Mao et al, ; Mullen et al, ). Labeling was visualized with the fluorescent secondary antibodies goat‐anti‐mouse Alexa Fluor 488 (Invitrogen) and goat‐anti‐rabbit Alexa Fluor 568 (Invitrogen).…”

Single-cell multimodal omics and directly reprogrammed neurons to probe reduced penetrance in Frontotemporal Dementia

“…6A) with antibody UMI levels (fig.4A) across all available cell types. The analysis suggests that NeuN antibody (mouse clone A60) used in our study shows unspecific binding [73] in the non-neuronal cell types. Typically, in FTD associated with GRN mutation, cortical layers 2/3 are affected by the loss of neurons [61] and downregulation of NeuN levels [137].…”

Reduced Granule Cell Proliferation and Molecular Dysregulation in the Cerebellum of Lysosomal Acid Phosphatase 2 (ACP2) Mutant Mice

“…Briefly, the sections were washed with PBS and treated with 0.3% H 2 O 2 to stop the endogenous peroxidase activity, followed by another wash. Sections were kept in blocking solution (10% normal goat serum and 0.05% Triton x-100 in PBS) for 1 h and subsequently incubated overnight at room temperature with primary antibody (anti-NeuN rabbit polyclonal, Millipore Sigma, Oakville, ON, Canada, 1:1000 [ 72 ]; anti-PAX6 mouse monoclonal, Developmental Studies Hybridoma Bank [ 73 ], 1:25; anti-calbindin D-28K mouse monoclonal, Swant, 1:1000 [ 39 ]; anti-sonic hedgehog rabbit polyclonal, Millipore Sigma, 1:200 [ 19 ]; anti-MYCN mouse monoclonal, Millipore Sigma, 1:25 [ 74 ], anti-proteasome 20S alpha + beta rabbit polyclonal, Abcam, Toronto, ON, Canada, 1:400, and anti-GFAP rabbit polyclonal (Millipore Sigma, 1:1000). After thorough washing with PBS, sections were treated for 2 h with the appropriate secondary antibody (goat anti mouse IgG, HRP conjugate, Millipore Sigma, 1:500; goat anti rabbit IgG, HRP conjugate, Millipore Sigma, 1:500).…”