Neuronal expression was assessed using a neuron-specific marker, NeuN antibody (1∶500 dilution, Chemicon), which is a DNA binding protein that will bind to the nucleus of neurons. Activated microglia and brain macrophages were observed using the CD11b antibody (1∶1000 dilution, Serotec) which is commonly known as OX42 and used to visualise activated microglia [21] (link), [22] . Frozen coronal cryostat sections (16 µm) were post-fixed in paraformaldehyde (4%). Slides were incubated overnight with either the NeuN or OX42 antibody at 4°C. Sections were then incubated at room temperature with a fluorescence labelled secondary antibody, Alexa 488 (1∶500 dilution, Invitrogen). The number of immuno-positive cells were counted within a 1 mm2 site for the NeuN antibody and a 0.5 mm2 site for the OX42-immuno staining in the infarcted region of the ipsilateral hemisphere. Ballistic light images from consecutive sections for each individual rat were used to locate the infarct and non-infarct regions in which cell counts were conducted. As a control, the number of immuno-positive cells in the identical region in the contralateral hemisphere was also counted. In addition, the number of immuno-positive cells in within the non-infarcted region of the ipsilateral hemisphere and matched region on the contralateral hemisphere was also calculated.
Neun antibody
Manufactured by Merck Group
Sourced in United States, United Kingdom
The NeuN antibody is a laboratory reagent used to detect the presence of the NeuN protein, which is a neuronal nuclear protein commonly used as a marker for identifying mature neurons in various tissues. The antibody binds specifically to the NeuN protein, allowing for the visualization and quantification of neurons in biological samples.
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Lab products found in correlation
Gfap antibody, by Merck Group (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Real time pcr rt pcr primers, by Qiagen (3 mentions)
Abc elite, by Vector Laboratories (2 mentions)
Confocal laser scanning microscope, by Leica camera (2 mentions)
Cy3 labeled anti rat igg antibody, by Merck Group (2 mentions)
Fluorescein labeled anti mouse antibody, by Merck Group (2 mentions)
Fitc labeled anti mouse antibody, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Antibodies against iba1, by Fujifilm (2 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Cd11b antibody, by Bio-Rad (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Mouse monoclonal anti brdu antibody, by Roche (1 mentions)
Biotinylated mouse secondary antibody, by Vector Laboratories (1 mentions)
Permount mounting media, by Thermo Fisher Scientific (1 mentions)
P stat3 antibody, by Cell Signaling Technology (1 mentions)
Iba 1 antibody, by Cell Signaling Technology (1 mentions)
45 protocols using neun antibody
1
Neuronal and Microglial Immunohistochemistry
2
Immunohistochemical Analysis of Mouse Brain and Spinal Cord
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Mice were deeply anesthetized and transcardially perfused with saline followed by fixative (4 % paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. Brain and spinal cord tissues were removed and post-fixed in the same fixative overnight. Following equilibration in 30 % sucrose in PBS, 30 μm sections were cut in the coronal plane with a cryostat at −20 °C and processed for immunohistochemistry. Primary antibodies against GFAP (1:1000, Sigma-Aldrich USA, St. Louis, MO), ionized calcium-binding adaptor molecule-1 (Iba1) (1:1000, Wako Chemicals USA, Richmond, VA), tyrosine hydroxylase (TH) (1:1000, Sigma-Aldrich USA, St. Louis, MO), α-syn (C20 & 211, 1:1000, Santa Cruz Biotech, Santa Cruz, CA), and secondary Alexa 488- or Alex 568-conjugated antibodies (1:1000, Invitrogen) were used to visualize the staining. Dapi (1:1000, Invitrogen) was used for counterstaining the nuclei. A monoclonal mouse anti-neuronal Nuclei (NeuN) antibody was used at a 1:1000 dilution (Chemicon International, Temecula, CA). Immunofluorescent images were captured using a laser scanning confocal microscope (Leica TCS SP8, Germany).
3
Quantifying Hippocampal Neuronal Proliferation
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For measuring proliferative neuronal cells in the hippocampal dentate gyrus, immunohistochemistry for BrdU was conducted with reference to the previously described method [13 (link),21 (link)]. First, the sections were permeabilized by incubation with 0.5% Triton X-100 in PBS for 20 minutes, then pretreated in 50% formamide-2X standard saline citrate for 2 hours at 65°C. The sections were denatured with 2N HCl for 30 minutes at 37°C, and rinsed twice with 100mM sodium borate (pH, 8.5). Then, the sections were incubated with mouse monoclonal anti-BrdU antibody (1:600; Roche, Mannheim, Germany) at 4°C overnight. The next day, the sections were washed with PBS 3 times and incubated with the mouse biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) for 120 minutes. Counter-staining was conducted on the same sections using a mouse monoclonal antineuronal nuclei (NeuN) antibody (1:1,000; Chemicon International, Temecula, CA, USA) following BrdU staining. After dehydration through a gradient of ethanol, the slides were mounted with coverslips using Permount mounting media (Thermo Fisher Scientific).
4
Neuronal Nuclei Immunostaining in Hippocampus
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Animals were sacrificed after 7 days of reperfusion that followed 10 min ischemia or ischemia + BK postconditioning. The nervous tissue sections were prepared by the same procedure on cryostat and randomly selected for immunocytochemistry, immunostaining for NeuN (Neuronal Nuclei, Neuron-Specific Nuclear Protein), a neuronal biomarker, in the CA1 region of hippocampus. The sections were incubated overnight at 4 °C with NeuN antibody (Chemicon International, Temecula, USA, 1:400) in 0.1 mol/l PBS (pH 7.4) with 0.2 % Triton. In the morning they were washed with 0.1 mol/l PBS (pH 7.4) with 0.2 % Triton, and the secondary anti-mouse IgG antibody was applied for 90 min at room temperature. The sections were washed again, and ABC Elite (Vector Laboratories, Burlingame, USA) was applied for 90 min. The slides were then rinsed with PBS followed by Tris Buffer (pH 7.6), and reacted with DAB (0.1 mol/l Tris, 0.04 % DAB, 0.033 % H2O2); the reaction was stopped with phosphate buffer. The slides were dehydrated, cleared and a coverslip applied for analysis.
5
Immunohistochemical Analysis of Cell Signaling
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Sections were treated with either p‐JAK2 antibody, p‐STAT3 antibody, cyclin D1 antibody (Cell Signaling Technology), CDK6 antibody (Cell Signaling Technology) with neuron‐specific nuclear protein (NeuN) antibody (Chemicon), glial fibrillary acidic protein (GFAP) antibody (Santa), or ionized calcium‐binding adapter molecule‐1 (Iba‐1) antibody (Cell Signaling Technology); either cyclin D1 antibody or CDK6 antibody with p‐STAT3 antibody at 4°C overnight. The sections were then incubated with a secondary antibody (Invitrogen) for 1 h at 22 ± 2°C. The nuclei were stained with DAPI.
For double staining of NeuN, GFAP, Iba‐1, cyclin D1, or CDK6 and TUNEL, slices were incubated with the primary antibodies against NeuN, GFAP, Iba‐1, cyclin D1, or CDK6, respectively, at 4°C overnight, with PBS used as a negative control. Following three washes in PBS, the sections were incubated with fluorescent‐labeled secondary antibodies. Sections were then stained using the TUNEL assay kit according to the manufacturer's instructions and counterstained with DAPI.
For double staining of NeuN, GFAP, Iba‐1, cyclin D1, or CDK6 and TUNEL, slices were incubated with the primary antibodies against NeuN, GFAP, Iba‐1, cyclin D1, or CDK6, respectively, at 4°C overnight, with PBS used as a negative control. Following three washes in PBS, the sections were incubated with fluorescent‐labeled secondary antibodies. Sections were then stained using the TUNEL assay kit according to the manufacturer's instructions and counterstained with DAPI.
6
Immunohistochemistry of Brain Slice Cultures
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Slice cultures were left attached on the Millicell membrane and stained as free-floating sections in 6-well plates. Cultures were first fixed with 4% paraformaldehyde in PBS (PAA, Austria) for 2 h at 4°C. After washing with cold PBS, slices were permeabilized by 0.4% TritonX-100/PBS for 90 min at RT. Slices were then blocked with 5% BSA for 2 h and afterwards incubated with primary antibody diluted in PBS for 2-3 days at 4°C. After washing with PBS, slices were incubated with secondary antibody for 2 days at 4°C. After washing, slices were mounted with Permafluor mounting solution (Beckman Coulter, Paris, France), cover-slipped and dried before imaging. The following primary antibodies were used: monoclonal anti-neuronal nuclei (NeuN) antibody (Chemicon International, Temecula, CA) (1:1000), pan-Tau antibody K9JA (Dako, Hamburg, Germany, Nr. A0024 (1:1000)); anti-Iba1 antibody (Wako Chemicals, Germany) (1:1000); anti-GFAP antibody (Cell signaling technology) (1:1000), anti-BrdU (GenTex) (1:1000), DCX (Cell Signalling) (1:500) and Ki67 (Abcam) (1:500). All fluorescent (goat anti-rabbit/mouse cyanine 2, 3 and 5)-labeled secondary antibodies were from Dianova (Hamburg, Germany) (1:1000).
7
Immunocytochemistry of Hippocampal Neurons
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Immunocytochemistry was performed on the prepared coronal free-floating 33 μm vibratome sections. Sections containing the hippocampus were immunostained for NeuN, a neuronal marker. Briefly, the sections were incubated overnight at 4°C with NeuN antibody (Chemicon International, Temecula, USA, 1:500) in 0.1 mol/l PBS (pH 7.4) with 0.2% Triton-X. After washing with 0.1 mol/l PBS (pH 7.4) with 0.2% Triton-X, the secondary anti-mouse IgG antibody was applied for 90 min at room temperature. After further washing, ABC Elite (Vector Laboratories, Burlingame, USA) was applied for 90 min, then the slides were rinsed with PBS (phosphate-buffered saline) followed by Tris buffer (pH 7.6), and reacted with DAB (diaminobenzidine) (0.1 mol/l Tris, 0.04% DAB, 0.033% H 2 O 2 ); the reaction was stopped with phosphate buffer. The slides were dehydrated, cleared, and coverslipped for analysis.
8
Multifaceted Cellular Immunolabeling Assay
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The sections were incubated with rat monoclonal anti-BrdU antibody (1:200; Abcam, Cambridge, UK), which was revealed using CY3-labeled anti-rat IgG antibody (1:200) and mouse monoclonal anti-neuronal nuclei (NeuN) antibody (1:500, Millipore), which was revealed using fluorescein-labeled anti-mouse antibody (1:50) or mouse monoclonal anti-GFAP antibody (1:200, Millipore), which was revealed using FITC-labeled anti-mouse antibody (1:50). The immunopositive cells/fibers were observed by a confocal laser-scanning microscope (Leica, Heidelberg, Germany).
9
TUNEL and NeuN Double Staining
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TUNEL staining was performed using Promega DeadEnd Kit. Briefly, sections were fixed with 4% paraformaldehyde. After equilibration, sections were incubated with TdT reaction mix for 1hr at 37°C. Stop the reaction with 2xSSC and then mount and analyze. For double staining with NeuN, before TUNEL staining, sections were incubated with NeuN antibody (Milipore) followed by Alexa Fluor 647- tagged secondary antibody.
10
Immunohistochemical Analysis of Brain Sections
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Brain sections were reacted with primary antibodies of mouse anti-neural nuclei antigen (NeuN) antibody (Millipore, 1:250), mouse anti-ED1 antibody (Millipore, 1:500), or mouse anti-human-specific nuclei antigen antibody (Millipore, 1:100) at 4 °C for 12 h. Subsequently, the secondary antibody of goat anti-mouse IgG-conjugated biotin (Millipore, 1:250) was added and reacted for 1 h at room temperature. Slides were then washed with 0.1 M PBS three times (5 min each), reacted with avidin-biotinylated complex (ABC kit, Vector) for 1 h, washed three times with 0.1 M PBS (5 min each), and developed with DAB. After staining, the slides were mounted with a mounting medium (Fisher Scientific SP15-500) and examined and photographed under an optical microscope.
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