Anti‐cGAS (Cell Signalling, 15102, 1:1000); anti‐STING (Cell Signalling, 13647, 1:1000); anti‐pSTING (Cell Signalling, 40818, 1:200); anti‐Tubulin (Sigma‐Aldrich, T5168, 1:2000); anti‐p21 (Cell Signalling, 2946S, 1:400); anti‐Ki67 (ThermoFisher Scientific, PA1‐21520, 1:100); anti‐NeuN (Millipore, ABN78, 1:1000); anti‐NeuN (Millipore, MAB377, 1:1000); anti‐GFAP (Sigma‐Aldrich, G3893, 1:1000); anti‐GFAP (Agilent, Z0334, 1:2000); anti‐pNF‐κB (Cell Signalling, 3033, 1:500); anti‐pIRF3 (Cell Signalling, 37829, 1:400); anti‐JNK (R&D Systems, AF1387SP, 1:1000); anti‐NG2 (Sigma‐Aldrich, AB5320, 1:200); anti‐Mouse IgG (Jackson ImmunoResearch, 715‐165‐151, 1:500); anti‐rat IgG (Jackson ImmunoResearch, 712‐605‐153, 1:500); anti‐rabbit IgG (Jackson ImmunoResearch, 711‐165‐152, 1:500); HRP anti‐Mouse IgG (Cell Signalling, 7076S, 1:10000); HRP anti‐rabbit IgG (Cell Signalling, 7074S, 1:10,000).
Anti neun
Manufactured by Merck Group
Sourced in United States, Germany, Canada, United Kingdom, China, Japan, Sweden, Italy
Anti-NeuN is a primary antibody used for the detection of neuronal nuclei in immunohistochemical and immunocytochemical applications. It is a specific marker for mature neurons.
Automatically generated - may contain errors
Lab products found in correlation
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Anti map2, by Merck Group (13 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (12 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (11 mentions)
Anti gfap, by Abcam (10 mentions)
Anti iba1, by Abcam (8 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Anti brdu, by Abcam (7 mentions)
Anti gfap, by Thermo Fisher Scientific (7 mentions)
Anti gfap, by Cell Signaling Technology (6 mentions)
Anti nestin, by Merck Group (6 mentions)
Anti β actin, by Merck Group (6 mentions)
Alexa 488, by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Triton x 100, by Merck Group (6 mentions)
Anti gfap, by Santa Cruz Biotechnology (5 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (5 mentions)
266 protocols using anti neun
1
Comprehensive Antibody Validation Protocol
2
Immunohistochemical Analysis of Brain Sections
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Anesthetized mice were transcardially perfused with ice-cold PBS and 4% PFA. The brains were isolated, and regions of interest were dissected using a brain matrix, cryoprotected in sucrose 30%, and frozen in OCT. Frontal and sagittal sections were cut at a 20 μm thickness with a cryostat (Leica). Sections were blocked with 10% normal goat serum for 30 minutes at room temperature and then incubated with primary antibody anti-NeuN (1:1000, Chemicon) overnight at 4°C. Slides were then incubated with secondary antibody biotin-conjugated goat anti-mouse (KPL) for 1 hour at room temperature and Streptavidin-Peroxidase (KPL) for 30 minutes at room temperature. Staining was visualized using a solution of 0.05% 3,3′-diaminobenzidine, 50 mM Tris-HCl, pH 7.2, 0.02% H2O2. Images were captured with an optic microscope.
For immunofluorescence staining, sections were blocked with 10% normal goat serum for 1 hour at room temperature and permeabilized with 1% Triton X-100. Sections were incubated with primary antibody (GFAP, 1:500, Cell Signaling Technology; anti-NeuN, 1:1000, Chemicon) for 16 hours at 4°C. Slides were then incubated with Alexa Fluor secondary antibody for 1 hour at room temperature and mounted with Vectashield mounting medium (Vector Laboratories) for fluorescence. Images were captured with an Olympus BX51 confocal microscope.
For immunofluorescence staining, sections were blocked with 10% normal goat serum for 1 hour at room temperature and permeabilized with 1% Triton X-100. Sections were incubated with primary antibody (GFAP, 1:500, Cell Signaling Technology; anti-NeuN, 1:1000, Chemicon) for 16 hours at 4°C. Slides were then incubated with Alexa Fluor secondary antibody for 1 hour at room temperature and mounted with Vectashield mounting medium (Vector Laboratories) for fluorescence. Images were captured with an Olympus BX51 confocal microscope.
3
Immunohistochemical Analysis of Neuronal Markers
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Mice were killed after the behavioral tests. Mice from each group were deeply anesthetized with chloral hydrate and perfused transcardially with ice-cold 0.9% saline, followed by 4% paraformaldehyde. The brains were dissected from the skulls, post-fixed with 4% paraformaldehyde over night, followed by 10%, 20% and 30% sucrose solutions, each for at least 16 hours. Brain tissue was embedded in Tissue Freezing Medium (Leica, Germany), frozen at −80°C and cut with a Leica microtome into 20-μm coronal sections. Frozen sections were used to observe the expression of MBP, KLC and NF200. Neuronal status in the hippocampus was determined by NeuN immunofluorescence. Sections were incubated over night at 4°C with primary antibodies: anti-MBP (anti-rat monoclonal; 1:200; Abcam), anti-KLC (anti-rabbit monoclonal; 1:50; Abcam), anti-NF200 (anti-rabbit polyclonal; 1:200; Sigma), anti-NeuN (anti-rabbit polyclone; 1:200; EMD millipore). Following the incubation with primary antibodies, sections were washed and incubated for 2 h at room temperature with secondary antibody: donkey Alexa Fluor 488 F(ab) anti-rat IgG, goat Alexa Fluor 488 F(ab) anti-rabbit IgG, or goat Alexa Fluor 592 F(ab) anti-rabbit IgG. Images were captured from stained frozen sections using a fluorescence microscope.
4
Immunohistochemical Analysis of DNMT3a in Spinal Cord
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The lumbar spinal cord tissues were postfixed with 4% paraformaldehyde for 12 hours, cryoprotected by immersion in 30% sucrose solution overnight at 4°C, and sectioned on a freezing microtome set at 30 μm. Following an initial background block using 10% normal donkey serum/PBS–Triton X-100 at room temperature for 2 hours, the sections were then incubated with polyclonal primary rabbit anti-DNMT3a antibody (1:50; EpiGentek) in a humidity chamber for 24 hours at 4°C. After several rinses in 1 M PBS, the slides were subsequently reacted with secondary antibody, Alexa Fluor 594-conjugated anti-rabbit IgG (1:300; Thermo Fisher Scientific) for 2 hours at 37°C. For double immunolabeling, we used the following antibodies: mouse monoclonal antibody against neuron-specific nuclear protein (anti-NeuN; 1:500; EMD Millipore, Billerica, MA, USA) and Alexa Fluor 488-conjugated anti-mouse IgG (1:300; Thermo Fisher Scientific). After washing, the sections were mounted with Shandon PermaFluor (Thermo Fisher Scientific) and analyzed using a confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan). Specificity of staining was confirmed by omission of the primary antibody or the biotinylated secondary antibody in multiple sections. No positive staining was detected in any of these negative control experiments.
5
TUNEL Staining and NeuN Labeling
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TUNEL staining was detected by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) detection kit (Roche Inc., Indianapolis, USA) in line with the manufacturer's instructions. The brain slices and coverslips were incubated with the primary antibody anti-NeuN (1 : 200, EMD Millipore) overnight. And then, a reaction solution and converter-AP were incubated subsequently. The fluorescently stained cells were recorded using Image J program.
6
Immunostaining of Brain Tissue
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The PFA‐fixed slices were cut off from the membrane and transferred into 3% BSA solution for blocking. Afterward, the slices were incubated with primary antibodies (anti‐NeuN, Merck Millipore, Darmstadt, Germany and anti‐Iba1, Wako Chemicals GmbH, Neuss, Germany) overnight at 4°C. After the tissues were washed three times for 20 minutes, fluorescently labeled secondary antibodies (all Invitrogen, Karlsruhe, Germany; anti‐mouse IgG (H+L) AlexaFluor488, anti‐mouse IgG (H+L) AlexaFluor 546, anti‐rabbit IgG (H+L) AlexaFluor647, and anti‐rabbit IgG (H+L) AlexaFluor546, all 1:1000) were applied, and the slices were incubated for 2 h at room temperature. Again, after the slices were washed three times for 20 minutes each, the slices were transferred onto object slides and covered with fluorescence mounting medium (Dako, Santa Clara, USA), and a coverslip was placed on top.
7
Immunohistochemical Analysis of Circadian Clock Protein in Mouse Brain
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Brains were harvested after mice were cardiovascularly perfused with saline and 4% PFA, and the tissue was left in PFA over night, and then transferred to 30% sucrose in PBS for two days for cryoprotection. 40 µm sections were cut using a cryostat and kept at -20 °C in an antigen preservation solution (1% m/m polyvinyl pyrrolidone in a 1 : 1 mixture of PBS and ethylene glycol) until subsequent steps of washing, blocking with permeabilization, antigen retrieval and antibody staining (anti-PER2, Alpha Diagnostic, cat. PER21-A, 1 : 200 dilution; anti-GFAP, Abcam, cat. ab53554, 1 : 500 dilution; anti-NeuN, Merck Millipore, cat. mab377, 1 : 250 dilution). The procedures have been described by our laboratory in detail elsewhere9 (link). Images were acquired using a Leica SP5 confocal microscope.
8
Immunofluorescence Staining of Rat Brain
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Samples were collected from animals at 24 h after perforation for double-fluorescence staining, performed as previously described [18 (link)]. The steps of cells immunofluorescence refer to the experimental method reported by Elliot H Choi et al. [23 (link)]. In brief, rat brains were perfused with 0.9 % saline first, and next the 4% paraformaldehyde, then fixed with 4% paraformaldehyde, dehydrated, and frozen at −80 °C. The samples were then sliced into 10 μm thick frozen sections, which were exposed O/N at 4 °C to the individual primary antibodies as follows: anti-CNPase (1:50, Abcam, ab6319), anti-Iba-1 (1:50, Abcam, ab15690), anti-GFAP (1:50, Abcam, ab10062), anti-myelin basic protein (MBP, 1:50, Abcam, ab62631), anti-Dexras1 (1:50, Abcam, ab78459), anti-NG2 (1:50, Biorbyt, orb382135), anti-NEUN (1:50, Merck Millipore, Burlington, MA, USA). The appropriate secondary antibody (Proteintech, SA00003-1/SA00009-2, Wuhan, China) was incubated with the brain sections at RT for 2 h, and the sections were observed and photographed using a fluorescence microscope (FV1200, Olympus, Tokyo, Japan). The main brain region we looked at was the subcortical area of the left cerebral hemisphere that was enriched for Dexras1 after subarachnoid hemorrhage.
9
Immunocytochemistry of NeuN in SVZ cells
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SVZ cultures were fixed with 4% PFA seven days after being transfected. Cells were permeabilized and blocked for non-specific binding sites in PBS with 0.25% Triton X-100 and 6% BSA for 1 h. Cells were subsequently incubated overnight at 4 °C with a primary mouse monoclonal anti NeuN (1:100, Merck Millipore, Darmstadt, Germany) prepared in PBS with 0.3% BSA and 0.1% Triton X-100. Cells were then incubated for 1 h with the secondary antibody Alexa Fluor 546 donkey anti mouse (1:200, Life Technologies) followed by Hoechst-33342 nuclear staining and mounted in Fluoroshield Mounting Medium (Abcam Plc.). Photomicrographs were taken using an AxioImager microscope (Carl Zeiss).
10
Quantification of Abnormal Cerebellar Lesions
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The collected organs were formalin-fixed and paraffin-embedded (FFPE) for routine histology. To assess the percentage of PNLs, the entire cerebellum was examined, recovering FFPE brain sections with intervals of 70 μm. At the recognition of abnormal cerebellar regions—in general, defined as PNLs—sections were collected and stained with hematoxylin and eosin (H&E). PNLs cross-sectional areas were carried out using imaging software NIS-Elements BR 4.00.05 (Nikon Instruments Europe B.V., Campi Bisenzio, Italy).
For immunohistochemistry, PNL-positive FFPE brain sections (4 μm) were dewaxed for 20 min at 56 °C and incubated in citrate buffer pH 6.0 for 20 min at 95 °C. After peroxidases inhibition by 3% H2O2 for 10 min, sections were incubated with primary antibodies anti-Ki67 (Bethyl, Montgomery, TX, USA), anti-NeuN (Merck Millipore, Darmstadt, Germany), and anti-caspase-3-activated (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature in a moist chamber. After incubation with the secondary anti-rabbit antibody (Bethyl), the antigen–antibody reaction was revealed by DAB (Dako, Agilent Technologies, Santa Clara, CA, USA) and analyzed by HistoQuest (TissueGnostics, Vienna, Austria) software.
For immunohistochemistry, PNL-positive FFPE brain sections (4 μm) were dewaxed for 20 min at 56 °C and incubated in citrate buffer pH 6.0 for 20 min at 95 °C. After peroxidases inhibition by 3% H2O2 for 10 min, sections were incubated with primary antibodies anti-Ki67 (Bethyl, Montgomery, TX, USA), anti-NeuN (Merck Millipore, Darmstadt, Germany), and anti-caspase-3-activated (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature in a moist chamber. After incubation with the secondary anti-rabbit antibody (Bethyl), the antigen–antibody reaction was revealed by DAB (Dako, Agilent Technologies, Santa Clara, CA, USA) and analyzed by HistoQuest (TissueGnostics, Vienna, Austria) software.
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