Neun clone a60

Manufactured by Merck Group

Sourced in United States

NeuN (clone A60) is a lab equipment product offered by Merck Group. NeuN is a neuronal nuclear protein that is widely used as a marker for identifying mature neurons. It serves as a core function to detect and identify neuronal cell populations in various research and diagnostic applications.

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Lab products found in correlation

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Spelling variants of this product

Clone A60 (11 citations) Anti-NeuN (clone A60; (5 citations) NeuN; A60), (2 citations) Mouse anti-NeuN (clone A60, (2 citations)

6 protocols using neun clone a60

Facial Nerve Injury and Neuronal Responses

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Unilateral facial nerve transection at the stylomastoid foramen was performed in WT, ApoE^−/−, Trem2^−/−, and Cx3cr1^CreERT2:ApoE^fl/fl and Cx3cr1^wt:ApoE^fl/fl (control) mice (n = 4–5/group). Successful nerve injury indicated by ipsilateral whisker paresis was assessed upon recovery from mild anesthesia by isoflurane. All mice were sacrificed at 7 days post-facial nerve axotomy. Immunohistochemical staining for NeuN (clone A60, Millipore, 2 μg ml⁻¹) and P2ry12 (polyclonal, 0.4 μg ml⁻¹) and analyses were performed on 30 μm floating sections as described in previous section. For quantification of NeuN⁺ neurons, only cells within the facial motor nucleus with fully visible cell bodies and visible cytoplasm were included.

Krasemann S., Madore C., Cialic R., Baufeld C., Calcagno N., El Fatimy R., Beckers L., O’Loughlin E., Xu Y., Fanek Z., Greco D.J., Smith S.T., Tweet G., Humulock Z., Zrzavy T., Conde-Sanroman P., Gacias M., Weng Z., Chen H., Tjon E., Mazaheri F., Hartmann K., Madi A., Ulrich J., Glatzel M., Worthmann A., Heeren J., Budnik B., Lemere C., Ikezu T., Heppner F.L., Litvak V., Holtzman D.M., Lassmann H., Weiner H.L., Ochando J., Haass C, & Butovsky O. (2017). The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity, 47(3), 566-581.e9.

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Multimarker Immunohistochemistry for Tissue Analysis

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Tissues were fixed in 4% PBS-buffered paraformaldehyde overnight and then transferred to 30% sucrose (w/v) for another 24 hours. Tissue was then embedded in Optimal Cutting Temperature (OCT) compound and sectioned at 8 microns on a Leica cryostat. Sections were blocked (PBS, 5% goat serum, 0.1% TritonX-100), incubated with primary antibodies for 2 hrs at room temperature in humidity chamber, washed with PBS and incubated with secondary antibodies for 45 min room temperature. Primary antibodies used: GFP (1∶250; Aves Labs Cat# GFP-1020 RRID:AB_10000240), Gabra6 (1∶100; EMD Millipore Cat# AB5610 RRID:AB_91935), NeuN Clone A60 (1∶100; Millipore Cat# MAB377 RRID:AB_2298772), BLBP (1∶250; EMD Millipore Cat# ABN14 RRID:AB_10000325), Calb1 D28K (1∶100; Sigma-Aldrich Cat# C9848 RRID:AB_476894), Olig2 (1∶150; EMD Millipore Cat# AB9610 RRID:AB_570666), Ki67 (1∶100; Abcam Cat# ab15580 RRID:AB_443209), Rspo1 (1∶1000; Sigma-Aldrich Cat# SAB3500046 RRID:AB_10602510), Pax6 (1∶250; Covance Cat# PRB-278P), Prominin-1 (1∶100; eBioscience Cat# 14-1331-90), Nestin (1∶300; BD Biosciences Cat#556309). Species-specific Alexa-Fluor-conjugated secondary antibodies were used for detection (1∶250; Invitrogen). tdTomato expression was visualized directly.

Miller T.E., Wang J., Sukhdeo K., Horbinski C., Tesar P.J., Wechsler-Reya R.J, & Rich J.N. (2014). Lgr5 Marks Post-Mitotic, Lineage Restricted Cerebellar Granule Neurons during Postnatal Development. PLoS ONE, 9(12), e114433.

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Immunofluorescent Staining of Neural Markers

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Immunofluorescent staining was performed as described previously [15 (link)]. Primary antibodies used include: EphA3 C19 (Santa Cruz), EphA2 clone D7 (Millipore, Billerica, MA), NeuN clone A60 (Millipore), GFAP (Santa Cruz), CD31 (Pierce, Rockford, IL), CD68 clone SPM281 (Novus Biologicals, Littleton, CO), CD163 clone 5C6FAT (Novus Biologicals), Nestin (1:200, Santa Cruz) and CD206 clone 15-2 (Santa Cruz).

Ferluga S., Tomé C.M., Herpai D.M., D'Agostino R, & Debinski W. (2016). Simultaneous targeting of Eph receptors in glioblastoma. Oncotarget, 7(37), 59860-59876.

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Histopathological Characterization of CNS Tumors

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All samples with available tissue (n = 24/30) were histopathologically re-assessed according to the WHO 2016 classification of tumors of the central nervous system. Formalin-fixed, paraffin-embedded (FFPE) tissue samples were stained with hematoxylin and eosin (H&E) according to standard protocols. For all cases with sufficient material, immunohistochemistry was performed on a Ventana BenchMark ULTRA Immunostainer using either the OptiView DAB IHC Detection Kit or the ultraView Universal DAB Detection Kit (Ventana Medical Systems, Tucson, Arizona, USA). Antibodies were directed against: glial fibrillary acid protein (GFAP; Z0334, rabbit polyclonal, 1:1000 dilution, Dako Agilent, Santa Clara, CA, USA), Olig2 (clone EPR2673, rabbit monoclonal, 1:100 dilution, Abcam, Cambridge, UK), Synaptophysin (clone MRQ-40, rabbit monoclonal, 1:160 dilution, Cell Marque Corp., Rocklin, CA, USA), NeuN (clone A60, mouse monoclonal, 1:100 dilution, Millipore, Burlington, MA, USA), CD34 (clone QBEnd/10, mouse monoclonal, Ventana Medical Systems), Ki-67 (clone MIB-1, mouse monoclonal, 1:100 dilution, Dako Agilent).

Sievers P., Appay R., Schrimpf D., Stichel D., Reuss D.E., Wefers A.K., Reinhardt A., Coras R., Ruf V.C., Schmid S., de Stricker K., Boldt H.B., Kristensen B.W., Petersen J.K., Ulhøi B.P., Gardberg M., Aronica E., Hasselblatt M., Brück W., Bielle F., Mokhtari K., Lhermitte B., Wick W., Herold-Mende C., Hänggi D., Brandner S., Giangaspero F., Capper D., Rushing E., Wesseling P., Pfister S.M., Figarella-Branger D., von Deimling A., Sahm F, & Jones D.T.W. (2019). Rosette-forming glioneuronal tumors share a distinct DNA methylation profile and mutations in FGFR1, with recurrent co-mutation of PIK3CA and NF1. Acta neuropathologica, 138(3).

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Immunofluorescence Staining of Astrocytes and Neurons

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Sections were prepared as desribed above for GFAP staining. Primary antibodies α2 (Merck Millipore) diluted 1:300 and NeuN (clone A60, Merck Millipore) diluted 1:300 [49 (link)] were applied in 1% donkey serum PBS with 0.02% Triton X-100 overnight at 4 °C. The following day, secondary labeling was perfomred with Alexa Fluor^® 488-conjugated donkey anti-rabbit antibody and Alexa Fluor^® 568-conjugated streptavidin (Life Technologies) diluted 1:350 in 1% donkey serum PBS with 0.025% Triton X-100 for 1 h at room temperature. Hoechst (1:10,000) (Life technologies) was used to counterstain the nuclei. Sections were then mounted with fluorescence mounting medium (Dako) and subsequently analysed on a LSM510 laser-scanning confocal microscope using a 40× C-Apochromat water immersion objective NA 1.2 (Carl Zeiss). Zen 2011 software (Carl Zeiss) was used for image capturing and analysis.

Ellman D.G., Isaksen T.J., Lund M.C., Dursun S., Wirenfeldt M., Jørgensen L.H., Lykke-Hartmann K, & Lambertsen K.L. (2017). The loss-of-function disease-mutation G301R in the Na+/K+-ATPase α2 isoform decreases lesion volume and improves functional outcome after acute spinal cord injury in mice. BMC Neuroscience, 18, 66.

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Quantitative Immunohistochemistry of Neuronal Markers

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Coronal brain sections, 5 µm thick and mounted onto glass slides (three sections per slide), were deparaffinised, boiled for antigen retrieval in 10mM citric buffer and then incubated in 0.3% (v/v) hydrogen peroxidase solution. Sections were blocked for 20 min in blocking solution (0.1% (w/v) BSA in PBS), incubated in primary antibody (NeuN Clone A60, Merck Millipore, Burlington, MA, USA, diluted 1:11,000), followed by incubation in biotinylated goat anti-mouse IgG secondary antibody (Dako Denmark, Glostrup, Denmark). Primary and secondary antibodies were diluted in blocking solution and reactions were conducted for 1 h at room temperature (RT). Sections were then developed with diaminobenzidine solution (Dako Denmark) and embedded in Neo-Mount (Merck Millipore, USA). Images were taken using a light microscope at a 200× magnification (Carl Zeiss, Jena, Germany) and quantification was conducted by an investigator, blinded with respect to the genotype and the treatment. Neurons expressing NeuN were counted manually in a fixed area in hippocampal CA1, CA3 and dentate gyrus, all at Bregma level −3.16 ± 0.36, as well as in primary motor cortex (MC) at Bregma level 0.74 ± 0.36 [62 ].

Schwab K., Melis V., Harrington C.R., Wischik C.M., Magbagbeolu M., Theuring F, & Riedel G. (2021). Proteomic Analysis of Hydromethylthionine in the Line 66 Model of Frontotemporal Dementia Demonstrates Actions on Tau-Dependent and Tau-Independent Networks. Cells, 10(8), 2162.

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