Neuronal nuclei (neun)

Manufactured by Proteintech

Sourced in China, United States

NeuN is a DNA-binding, neuron-specific protein that is expressed in the nuclei of most neuronal cell types. It is commonly used as a marker for identifying and quantifying neurons in various tissues and experimental systems.

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19 protocols using neuronal nuclei (neun)

Immunofluorescence Assay for Neuronal Markers

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The immunofluorescence assay was performed as previously reported (Yang et al., 2015 (link)). Briefly, after fixation in 4% paraformaldehyde/phosphate‐buffered saline (PBS) for 10 min at room temperature (RT) and permeabilized with 0.3% Triton X‐100/PBS for 5 min. After blocking with 1% bovine serum albumin (BSA)/PBS for 20 min, the cells were incubated with primary antibodies, including Cav‐1 (1:100; Proteintech), HIF‐1α (1:100; Proteintech), NeuN (1:100; Proteintech), and Caspase3 (1:100; Proteintech), at 4°C overnight. After three washes, the cells were incubated with anti‐mouse or anti‐rabbit fluorescent secondary antibodies (1:200; Jackson Lab) at RT for 1 h. The nuclei were stained with Hoechst 33342 (1:1000; Sigma) for 20 min. Photos were obtained using a Leica microscope (Germany).
Brain slices (4–6 μm) from NMR and mouse brains were permeabilized with 0.1% Triton X‐100 for 10 min and blocked with 1% BSA/PBS for 20 min at RT. For immunofluorescence labelling studies, a variety of primary antibodies were applied, including Cav‐1 (1:100; Proteintech), NeuN (1:100; Proteintech), Caspase3 (1:100; Proteintech), and HIF‐1α (1:100; Proteintech), and fluorescent secondary antibodies (1:200; Jackson Lab) at RT for 1 h. Photos were obtained at RT using a Leica microscope (Germany).

Yang W., Wu W., Zhao Y., Li Y., Zhang C., Zhang J., Chen C, & Cui S. (2022). Caveolin‐1 suppresses hippocampal neuron apoptosis via the regulation of HIF1α in hypoxia in naked mole‐rats. Cell Biology International, 46(12), 2060-2074.

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Immunofluorescence Staining of Tissue Sections

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The obtained tissue Section (4 μm thickness) were incubated with primary antibodies against DCFH-DA (Beyotime Biotechnology, catalog number S0033S) or NeuN (Proteintech, catalog number 26975-1-AP) overnight at 4 °C. The sections were further coincubated with rabbit fluorescence secondary antibody for 120 min at room temperature. Finally, the stained sections were observed and photographed by confocal microscopy.

Xu L., Sun Z., Xing Z., Liu Y., Zhao H., Tang Z., Luo Y., Hao S, & Li K. (2022). Cur@SF NPs alleviate Friedreich’s ataxia in a mouse model through synergistic iron chelation and antioxidation. Journal of Nanobiotechnology, 20, 118.

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Quantifying Protein Expression in Spinal Cord

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Spinal cord samples were homogenized in a standard homogenizing buffer (50 mM Tris-HCl, pH 7.4; 5 mM EGTA; 1 mM phenylmethylsulfonyl fluoride) on ice plus protease inhibitor as described (Haque et al., 2007 (link); Haque et al., 2002 (link); O’Donnell et al., 2004 ). Equal protein concentrations from designated samples were separated on a 4–12% Bis/Tris NuPage gel (Invitrogen, Grand Island, NY) (God et al., 2015 ; Haque et al., 2017a (link); Hathaway-Schrader et al., 2018 (link)). Proteins were transferred onto a nitrocellulose membrane (Pierce, Rockford IL), and probed with caspase-1 (1:500, Santa Cruz, sc-622), NeuN (1:100, Proteintech, Catalog # 26975–1-AP), and MBP (1:1000, Millipore, MAB384) antibodies. The secondary antibodies used were horseradish peroxidase conjugated anti-mouse (1:2000, Santa Cruz, sc-2005), and anti-rabbit (1:4000, Santa Cruz, sc-2004). Monoclonal antibody for β-actin (1:1000, Santa Cruz, sc-81178) was used as a protein loading control. Relative protein expression was assessed using Image J software (National Institutes of Health, Bethesda, MD) and expressed as relative density for each sample (Goldstein et al., 2008 (link); Radwan et al., 2012 (link); Zhao et al., 2011 (link)).

Polcyn R., Capone M., Matzelle D., Hossain A., Chandran R., Banik N.L, & Haque A. (2020). Enolase inhibition alters metabolic hormones and inflammatory factors to promote neuroprotection in spinal cord injury. Neurochemistry international, 139, 104788.

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Immunofluorescent Staining of Brain Tissue

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Tissue sections were prepared as described above. The sections were incubated at 4°C overnight with primary antibodies against the following proteins: NeuN (anti-rabbit, 1 : 50, Proteintech, Wuhan, China), GFAP (anti-rabbit, 1 : 1000, Servicebio, Wuhan, China), and Iba1 (anti-rabbit, 1 : 500, Servicebio, Wuhan, China). Then, the sections were incubated with CY3-labeled goat anti-rabbit (1 : 300, Servicebio, Wuhan, China) fluorescent secondary antibody at room temperature for 50 min. TUNEL staining was then carried out using an in situ cell death assay kit (Roche, Mannheim, Germany). Nuclei were counterstained using DAPI (Servicebio, Wuhan, China). The sections were observed under a fluorescence microscope (Olympus).

Deng Y., Yang L., Xie Q., Yang F., Li G., Zhang G., Li S., Wu Z., Wang J, & Kang X. (2020). Protein Kinase A Is Involved in Neuropathic Pain by Activating the p38MAPK Pathway to Mediate Spinal Cord Cell Apoptosis. Mediators of Inflammation, 2020, 6420425.

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Western Blotting of Nrf2, β-actin, and NeuN

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Western blotting was performed as described previously by our laboratory (Zhang et al., 2008 (link)). The protein from cytosolic and nuclear extracts was separated on 4–20% SDS-polyacrylamide gels, and transferred onto PVDF membrane. The membranes were blocked and incubated with primary antibody Nrf2 (1:500), β-actin (1:2000) and Neuronal nuclear antigen (NeuN, 1:500, Proteintech) at 4°C overnight and HRP-conjugated secondary antibodies (1:1000, Cell signaling) for 1 h at room temperature (RT). Protein bands were visualized with digital imaging system. The protein levels of each sample were normalized as intensity ratio with β-actin or NeuN and were analyzed by ImageJ software.

Zhang J., Tucker L.D., Yan D., Lu Y., Yang L., Wu C., Li Y, & Zhang Q. (2018). Tert-butylhydroquinone Post-treatment Attenuates Neonatal Hypoxic-ischemic Brain Damage in Rats. Neurochemistry international, 116, 1-12.

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Quantitative Protein Analysis in Zebrafish Brain

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Protein extraction and quantitative analysis were performed according to the supplier’s instructions. For frozen zebrafish whole brain samples (one sample contained approximately 30 mg of brain tissue from five zebrafish, BD, n = 3; control, n = 3), cytoplasmic and nuclear proteins were extracted from the samples using a protein extraction kit (Solarbio, Cat. #R0050). BCA protein assay kit (Beyotime, Cat. #P0011) was used to determine protein concentrations. Western blotting was performed using the Wes-Simple Western system, an automated capillary-based size-sorting system containing an anti-rabbit detection module for Wes and a Wes Separation Capillary Cartridges for 12 to 230 kDa (ProteinSimple, Cat. #PDM-001 +SM-W004). Protein expression was measured by chemiluminescence and quantified as the area under the peak of the chemiluminescence chromatogram using Compass (ProteinSimple, USA). Proteins were detected using the following primary antibodies: GFAP (Proteintech, Cat. #16825-1-AP), NeuN (Proteintech, Cat. #26975-1-AP), Iba-1 (Proteintech, Cat. #10904-1-AP), Synaptophysin (Abcam, Cat. #ab32594), β-Actin (Proteintech, Cat. #66009-1-IG), Anti-JNK1 + JNK2 + JNK3 (Abcam, Cat. #ab179461), Phospho-SAPK/JNK (Cell Signalling Technology, Cat. #4668T), p38 MAPK (Proteintech, Cat. #14064-1-AP) and Phospho-p38 MAPK (Cell Signalling Technology, Cat. #4511T).

Li Y., Zhang L., Mao M., He L., Wang T., Pan Y., Zhao X., Li Z., Mu X., Qian Y, & Qiu J. (2023). Multi-omics analysis of a drug-induced model of bipolar disorder in zebrafish. iScience, 26(5), 106744.

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Immunofluorescence-Based Cell Quantification

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Immunofluorescence staining was in accord with previous description (Li D. et al., 2016 (link)). After being blocked, coronal sections were incubated with anti-human nuclei antibody (MAB1281, 1:100, Millipore, Oxford, UK), myelin basic protein (MBP; 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), Ki67 (1:200, Bioss, Beijing), DCX (1:200, Proteintech, China), or NeuN (1:200, Proteintech, China) at 4°C overnight and then incubated in Cy3/FITC-conjugated anti-mouse/rabbit anti IgG (1:500, Proteintech, China) for 1 h at room temperature, followed by DAPI (1:2,000, Biotech, China) staining for 10 min. The slides were examined with a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany). Positive-cells were counted with ImageJ software.

Xu L., Xing Q., Huang T., Zhou J., Liu T., Cui Y., Cheng T., Wang Y., Zhou X., Yang B., Yang G.L., Zhang J., Zang X., Ma S, & Guan F. (2019). HDAC1 Silence Promotes Neuroprotective Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Mouse Model of Traumatic Brain Injury via PI3K/AKT Pathway. Frontiers in Cellular Neuroscience, 12, 498.

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Quantifying Synaptic Markers in Rat Hippocampus

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One day after the MWM test, we used a triple fluorescein-labeled method to detect rat hippocampal tissues (n = 7). Rat hippocampal slices were cut into 20-μm thick slices for immunofluorescence staining. The samples were blocked in PBS containing 0.3% Triton X-100 (Sigma-Aldrich, Munich, Germany) and 5% bovine serum album (Beyotime, Beijing, China) for 2 h at room temperature. The samples were then incubated with synapsin-I (Proteintech, 1:100, Cat No. 17785-1-AP), p-CREB (Cell Signaling Technology, 1:100, Cat. no. 9198S), and Neun (Proteintech, 1:100, Cat No. 26975-1-AP) overnight at 4°C. The sections were washed three times with PBS before being incubated for 1 h with a goat anti-rabbit immunoglobulin G-FITC secondary antibody (1:100, Beyotime, P0186). Finally, the sections were stained using 4’,6-diamidino-2-phenylindole (DAPI; Beyotime, P0131). The fluorescence microscopy images were then quantitatively analyzed using the Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, United States) using a Nikon Eclipse CI fluorescence microscope.

Li Y., Zhang Q., Yan W., Wang X., Yu J., Yin C., Zhou Q., Hou Z, & Wang Q. (2022). Young plasma reverses anesthesia and surgery-induced cognitive impairment in aged rats by modulating hippocampal synaptic plasticity. Frontiers in Aging Neuroscience, 14, 996223.

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Molecular Characterization of Neurodegeneration

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After treatment for 28 days, 6 mice in each group were sacrificed under anesthesia. Brain tissues around damaged areas were isolated and western blot were performed. In brief, tissues were lysed in lysis buffer. Then an equal amount of protein was loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). After that, primary antibodies against neuronal differentiation related proteins (NSE, NeuN, and NFL, Proteintech), neurotrophic factors (BDNF, Proteintech), inflammation-associated protein (IL-6, Proteintech) and apoptosis-related proteins (Bax, Bcl-2, Proteintech) were incubated respectively, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody. β-actin was used as an internal control. The protein analysis was visualized by using Quantity One software (Azure Biosystems C300, Azure c300, USA).

Wang L., Zhang D., Ren Y., Guo S., Li J., Ma S., Yao M, & Guan F. (2021). Injectable hyaluronic acid hydrogel loaded with BMSC and NGF for traumatic brain injury treatment. Materials Today Bio, 13, 100201.

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Immunohistochemical Analysis of Neural Apoptosis

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Brain paraffin sections (4 μm) were hydrated and Tris/EDTA buffer performed heat-mediated antigen retrieval for 20 min. The sections blocked with 5% BSA for 1 h were incubated with Neun (Proteintech) along with primary antibodies Caspase-1 (Novus) overnight at 4°C and subsequently incubated in fluorescent secondary antibodies (Proteintech) for 1 h at 24°C. DAPI (Antgene) was used for nuclei staining. Images were taken with an Olympus BX53 microscope (Olympus). Positive cells were counted using ImageJ software (n = 6/group).

Liu J., Zheng J., Xu Y., Cao W., Wang J., Wang B., Zhao L., Zhang X, & Liao W. (2021). Enriched Environment Attenuates Pyroptosis to Improve Functional Recovery After Cerebral Ischemia/Reperfusion Injury. Frontiers in Aging Neuroscience, 13, 717644.

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