NeuN antibody (NEUronal Nuclei; clone A60) specifically recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most CNS and PNS neuronal cell types of all vertebrates tested. NeuN protein distributions are apparently restricted to neuronal nuclei, perikarya and some proximal neuronal processes in both fetal and adult brain although, some neurons fail to be recognized by NeuN at all ages: INL retinal cells, Cajal-Retzius cells, Purkinje cells, inferior olivary and dentate nucleus neurons, and sympathetic ganglion cells are examples (Mullen et al., 1992; Wolf et al., 1996). Immunohistochemically detectable NeuN protein first appears at developmental timepoints that correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron (Mullen et al., 1992). Immunoreactivity appears around E9.5 in the mouse neural tube and is extensive throughout the developing nervous system by E12.5. Strong nuclear staining suggests a nuclear regulatory protein function; however, no evidence currently exists as to whether the NeuN protein antigen has a function in the distal cytoplasm or whether it is merely synthesized there before being transported back into the nucleus. No difference between protein isolated from purified nuclei and whole brain extract on immunoblots has been found (Mullen et al., 1992).
MILLIPORE′s exclusive monoclonal antibody to vertebrate neuron-specific nuclear protein called NeuN (or Neuronal Nuclei) reacts with most neuronal cell types throughout the nervous system of mice including cerebellum, cerebral cortex, hippocampus, thalamus, spinal cord and neurons in the peripheral nervous system including dorsal root ganglia, sympathetic chain ganglia and enteric ganglia. Developmentally, immunoreactivity is first observed shortly after neurons have become postmitotic, no staining has been observed in proliferative zones. The immunohistochemical staining is primarily localized in the nucleus of the neurons with lighter staining in the cytoplasm. The few cell types not reactive with MAB377 include Purkinje, mitral and photoreceptor cells. The antibody is an excellent marker for neurons in primary cultures and in retinoic acid-stimulated P19 cells. It is also useful for identifying neurons in transplants.
Purified cell nuclei from mouse brain
Western Blot Analysis:
A previous lot of this antibody recognized 2-3 bands in the 46-48 kDa range and possibly another band at approximately 66 kDa.
1:10-1:100 dilution from a previous lot was used. Neurons in culture should be permeablized with 0.1% triton X-100. All primary antibody dilutions should be performed with simple solutions containing only buffer and primary antibody without excess protein blocks or detergents.
1:100-1:1,000. The antibody works best on polyester wax embedded tissue but also works on paraffin embedded tissue at a lower working dilution. The antibody works well with formaldehyde-based fixatives. Citric acid and microwave pretreatment has been used successfully (Sarnat, 1998).
Immunohistochemistry(paraffin) Analysis: A previous lot was used for IH(P).
Optimal working dilutions must be determined by end user.
Research Sub Category
Anti-NeuN Antibody, clone A60 detects level of NeuN and has been published and validated for use in FC, IC, IF, IH, IH(P), IP and WB.
Routinely evaluated by immunohistochemistry on brain tissue.
NeuN (cat. # MAB377) staining pattern/morphology in rat cerebellum. Tissue pretreated with Citrate, pH 6.0. This lot of antibody was diluted to 1:100, using IHC-Select Detection with HRP-DAB. Immunoreactivity is seen as nuclear staining in the neurons in the granular layer. Note that there is no signal detected in the nucleus of Purkinje cells.
Optimal Staining With Citrate Buffer, pH 6.0, Epitope Retrieval: Rat Cerebellum
Purified mouse immunoglobulin IgG1 liquid in buffer containing 0.02 M phosphate buffer, 0.25 M NaCl, pH 7.6 with 0.1% sodium azide.
Protein A purified
Stable for 6 months at 2-8ºC from date of receipt.
Positive control -Brain Tissue. Negative control - Any non neuronal tissue eg Fibroblasts
Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.
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