The brains were perfused with 4% paraformaldehyde in PBS (pH 7.4) and frozen. Immunohistochemistry was performed on 30 µm sections using rabbit polyclonal anti-mouse Ngb (Sigma 1:100), mouse monoclonal anti-NeuN (Chemicon; 1:200), rat monoclonal anti-PDGFRβ (Abcam; 1:100), mouse monoclonal anti-αSMA (Abcam; 1:100), mouse monoclonal anti-glial fibrillary acidic protein (Sigma; 1:200), as primary antibodies, and Alexa Fluor 488-conjugated goat anti-rat IgG (Abcam; 1:500), Alexa Fluor 594-conjugated goat anti-mouse IgG (Invitrogen; 1:500), and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen; 1:500) as secondary antibodies. Controls included omitting primary or secondary antibodies. Fluorescence signals were detected using a Zeiss LSM 800 confocal laser scanning microscope at excitation/emission wavelengths of 495/519 (Alexa Fluor 488), 556/573 (Alexa Fluor 546), 590/617 (Alexa Fluor 594), and 358/461 (DAPI) nm. In order to quantify Ngb expressed around blood vessels, we measured Ngb signals within 2 mm of all blood vessels using 512×512-pixel figures by image J.
Mouse monoclonal anti neun
Manufactured by Merck Group
Sourced in United States
Mouse monoclonal anti-NeuN is a laboratory reagent used to detect the NeuN protein, which is a neuron-specific nuclear protein. It is commonly used as a marker for identifying and quantifying neuronal cells in various applications, such as immunohistochemistry and flow cytometry.
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Lab products found in correlation
Rabbit polyclonal anti iba1, by Fujifilm (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Polyclonal rabbit anti gfap, by Agilent Technologies (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Mouse monoclonal anti α sma, by Abcam (1 mentions)
Mouse monoclonal anti glial fibrillary acidic protein, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti rat igg, by Abcam (1 mentions)
Alexa fluor 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Lsm 800 confocal laser scanning microscope, by Zeiss (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Abcam (1 mentions)
Rabbit polyclonal anti gfp, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 goat anti rat igg, by Abcam (1 mentions)
Alexa fluor 594 donkey anti rabbit igg, by Abcam (1 mentions)
Ix81 zdc2, by Olympus (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Abcam (1 mentions)
34 protocols using mouse monoclonal anti neun
1
Quantifying Neuroglobin Expression Around Blood Vessels
2
Immunohistochemical Analysis of Cell Markers
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Immunohistochemical staining was performed as previously reported (Zhang et al., 2015). On day 7 or 21 following PCI, the brains were removed, and 5-µm frozen coronal sections were prepared. After antigen retrieval, sections were immunolabeled with rat monoclonal anti-BrdU (1:500; Accurate Chemical & Scientific), rabbit polyclonal anti-GFP (1:200; Santa Cruz Biotechnology), mouse monoclonal anti-NeuN (1:500; Chemicon), rabbit monoclonal anti-GFAP (1:200; Chemicon), mouse monoclonal anti-RIP (1:50; kind gift from Dr Xu XM, University of Louisville School of Medicine) and mouse monoclonal anti-NgR1 (1:200; Biogen Idec, Inc.) overnight at 4°C. After washing with PBS, the sections were incubated with goat anti-rabbit IgG Alexa Fluor 488, goat anti-rat IgG Alexa Fluor 488, goat anti-mouse IgG Alexa Fluor 594 and donkey anti-rabbit IgG Alexa Fluor 594 (1:1000; Abcam) at room temperature for 1 hour. Cells double-positive for BrdU/NeuN, GFP/GFAP and GFP/RIP were counted. Positive cells were also visualized using a motorized inverted microscope (IX81-ZDC2; Olympus, Hamburg, Germany).
3
Neuronal viability assessment after SCI
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Neuronal viability was evaluated using monoclonal antibody NeuN (
He et al., 2017 ). Sections from the lesion epicenter were collected at 35 days after SCI. The transverse sections (7 μm thick) were deparaffinized and rehydrated to the standard protocol. Then sections were blocked for 60 min in 5% Bovine Serum Albumin (BSA) (PBS containing 5% BSA and 0.2% Triton X-100) and incubated with the appropriate primary antibodies, mouse monoclonal anti-NeuN (a neuronal marker) (1:1000, Chemicon, USA) at 4 °C overnight. After washing with PBS, the secondary antibody (FITC -conjugated IgG) was added, and the sections were incubated at room temperature for 1 h. The sections were mounted, and fluorescent labeling was visualized and captured using a fluorescence microscope.
4
Western Blot Protein Analysis Protocol
5
Western Blot Analysis of Neuronal and Inflammatory Proteins
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Proteins were extracted from brain tissues (100 mg), BV-2 cells (1 × 105), and N2a cells (1 × 105), respectively, and were subjected to SDS-PAGE. Separated proteins were transferred onto nitrocellulose membranes (Millipore Corp, Billerica, MA) and were blocked with 5% skimmed milk. After blocking, the membranes were incubated with the primary antibodies, including mouse monoclonal anti-NeuN (1:1 000, Chemicon, Temecula, CA), mouse monoclonal anti-iNOS (1:500, BD, Bio-science), and mouse monoclonal anti-actin (1:500, Santa Cruz, CA). After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10 000, Zhongshan BioTech Co, China). The protein bands were visualized by using an ECL kit (Super Signal West Pico, Thermo Scientific, USA).
6
Immunohistochemical Characterization of Neurogenesis
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Sections from the whole right hemisphere of P2 mice and sections from the right hippocampal formation of adult mice were incubated with a primary rat anti-BrdU antibody (diluted 1:200; Biorad) and a mouse monoclonal anti-NeuN (Neuronal-specific nuclear protein), a marker of mature neurons (diluted 1:250, Chemicon, Billerica, MA, USA). Detection was performed using a Cy3-conjugated anti rat-secondary antibody for BrdU immunohistochemistry (diluted 1:200; Jackson Immunoresearch) and a FITC-conjugated anti-mouse IgG for NeuN immunohistochemistry (diluted 1:200; Jackson Immunoresearch). Sections were additionally incubated for 2 min in Hoechst nuclear dye (0.2 mg/ml in PBS). The penetration of the anti-BrdU antibody was checked through 3-D image reconstruction (see Supplementary Fig. 2 ).
7
Antibody Staining Protocol for c-Fos and Netrin-G1
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Primary antibodies used in this study included rabbit polyclonal antibodies for netrin-G112 (link), rabbit polyclonal anti-c-Fos (Ab-5, Calbiochem), rabbit polyclonal anti-GABA (Sigma), mouse monoclonal anti-NeuN (Chemicon), rabbit polyclonal anti-ß-galactosidase (MP Biomedicals), and mouse monoclonal anti-ß-galactosidase (Promega). The secondary antibodies used for immunofluorescence were Alexa Fluor 488 and 546 donkey anti-mouse or rabbit IgG (Molecular Probes). Biotinylated goat anti-rabbit IgG and a Vectastain ABC kit (Vector Laboratories) were used for c-Fos staining. Mice for c-Fos analysis were firstly exposed to EPM or FC training and then sacrificed and perfused 100 minutes later. The c-Fos staining was performed as described previously59 (link). Rabbit polyclonal antibodies against Netrin-G1 were used as described previously12 (link).
8
Immunohistochemical Analysis of Neurogenesis
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Frozen 30-μm-thick sections were incubated in blocking solution (10% normal donkey serum or 10% normal goat serum and 0.3% Triton X-100 in PBS, pH 7.5) for 2 h at room temperature (RT) and then incubated with primary antibodies in 5% serum and PBS for 24 h at 4°C. The sections were subsequently incubated for 3 h at RT with fluorophore-conjugated secondary antibodies. To prepare the sections for BrdU staining, they were incubated in 2 N HCl for 0.5 h at 37°C.
Rat monoclonal anti-BrdU (1:100; Abcam, UK) primary antibodies were used to mark proliferating cells, mouse monoclonal anti-Nestin (1:100; BD Pharmingen, USA) primary antibodies were used as a marker for NSCs, and mouse monoclonal anti-NeuN (1:100; Chemicon, USA) primary antibodies were used to label mature neurons. The following secondary antibodies were used: Alexa Fluor 594-labeled donkey anti-rat IgG and Alexa Fluor 488-labeled donkey anti-mouse IgG (1:200 for both; Invitrogen, USA).
The stained slides were dehydrated, cover-slipped in anti-quenching agent (p-phenylenediamine, PPD) and analyzed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan). The number of positive cells was counted in a blinded manner in the ipsilateral SGZ using 20X and 40X objectives in OLYMPUS FV10-ASW Viewer.
Rat monoclonal anti-BrdU (1:100; Abcam, UK) primary antibodies were used to mark proliferating cells, mouse monoclonal anti-Nestin (1:100; BD Pharmingen, USA) primary antibodies were used as a marker for NSCs, and mouse monoclonal anti-NeuN (1:100; Chemicon, USA) primary antibodies were used to label mature neurons. The following secondary antibodies were used: Alexa Fluor 594-labeled donkey anti-rat IgG and Alexa Fluor 488-labeled donkey anti-mouse IgG (1:200 for both; Invitrogen, USA).
The stained slides were dehydrated, cover-slipped in anti-quenching agent (p-phenylenediamine, PPD) and analyzed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan). The number of positive cells was counted in a blinded manner in the ipsilateral SGZ using 20X and 40X objectives in OLYMPUS FV10-ASW Viewer.
9
Immunostaining of Neurogenic Markers in Brain Tissue
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The immunostaining assays were performed using a protocol adapted from18 (link). First, brain sections were incubated with 2 M HCl for 25 min at 37 °C to induce DNA denaturation. After washing with PBS, tissue sections were further incubated in a blocking solution containing 2% of horse serum (Life Technologies, Carlsbad, CA, USA) and 0.3% Triton X-100 (Fisher Scientific) diluted in 0.1 M PBS for 2 h at RT. After the blocking procedure, tissue sections were incubated for 72 h at 4 °C in the following primary antibodies (diluted in the blocking solution): rat monoclonal anti-BrdU (1:500, AbD Serotec, Raleigh, NC, USA), goat polyclonal anti-doublecortin (DCX; 1:500, Santa Cruz Biotechnology), or mouse monoclonal anti-NeuN (1:500, Merck Millipore). Then, sections were rinsed in PBS and incubated with Hoechst (1:1000; Sigma-Aldrich Co. LLC) and the respective secondary antibodies: Alexa Fluor-488 donkey anti-rat, Alexa Fluor-546 donkey anti-goat or anti-mouse (all 1:500; all Life Technologies), diluted in a solution containing 0.3% Triton X-100 in 0.1 M PBS, for 2 h at RT. Finally, sections were rinsed in PBS and mounted in Fluoroshield Mounting Medium (Abcam Plc., Cambridge, UK) for further analysis.
10
Antibody Landscape for Cellular Analysis
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The following antibodies were purchased: rabbit polyclonal anti-Rnd2 from Proteintech (Rosemont, IL); mouse monoclonal anti-myelin basic protein (MBP), and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) from Covance (Princeton, NJ); mouse monoclonal anti–β-actin from Fujifilm (Tokyo, Japan); goat polyclonal anti–platelet-derived growth factor receptor (PDGFR) α from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal anti-MBP, mouse monoclonal anti–adenomatus polyposis coli (APC) (also called CC1), mouse monoclonal anti-O4, and mouse monoclonal anti-NeuN from Merck-Millipore (Billerica, MA); rabbit polyclonal anti–phosphorylated Thr696-Mbs and rabbit polyclonal anti-Mbs from Cell Signaling Technology (Danvers, MA); rabbit monoclonal anti-Olig2, rabbit polyclonal anti–phosphorylated Ser1366-Rho kinase, which recognizes active states, and rabbit polyclonal anti-Rho kinase from Abcam (Cambridge, UK); mouse monoclonal anti-2′, 3′-cyclicnucleotide 3′-phosphodiesterase (CNPase) and rabbit polyclonal anti-neurofilament (NF) large subunit from Sigma-Aldrich (St. Louis, MO); peroxidase-conjugated secondary antibodies from MBL (Nagoya, Japan); and fluorescence-labeled secondary antibodies from Thermo Fisher Scientific (Waltham, MA).
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