For immunofluorescence staining of serial brain coronal sections (12 μm) and cultured cells, the tissues and cells were blocked with 1% bovine serum albumin and 1% goat normal serum 1 h at room temperature. Sections and cells were incubated at 4 °C overnight with primary antibodies: mouse anti-KCa3.1 (1:100; Alomone Labs), rabbit anti-GFAP (1:500; Dako); rabbit anti-Iba1 (1:500; Abcam); rabbit anti-NeuN antibody (1:500; Millipore), rabbit anti-TRPV4 (1:200; Alomone Labs). The sections and cells were incubated with following secondary antibodies: Alexa Fluor 555 goat anti-rabbit IgG (1:500; Invitrogen), Alexa Fluor 488 goat anti-mouse IgG (1:500; Invitrogen) for 1 h at room temperature. Then washed with PBS and stained with DAPI (4′, 6-diamidino-2-phenylindole).
Rabbit anti neun antibody
Manufactured by Merck Group
Sourced in United States
The Rabbit anti-NeuN antibody is a laboratory reagent used to detect the Neuronal Nuclei (NeuN) protein, which is a widely expressed marker of mature neuronal cells. This antibody can be used in various immunohistochemical and immunocytochemical applications to identify and visualize neuronal cell populations.
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Lab products found in correlation
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gfap, by Agilent Technologies (1 mentions)
Rabbit anti iba1, by Abcam (1 mentions)
Mouse anti kca3, by Alomone (1 mentions)
Rabbit anti trpv4, by Alomone (1 mentions)
Mouse anti gfap antibody, by Merck Group (1 mentions)
Anti mouse cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Confocal microscope, by Leica (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Tunel apoptosis detection kit, by Roche (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Iba1 antibody, by Fujifilm (1 mentions)
Mouse anti nlrp3 antibody, by Thermo Fisher Scientific (1 mentions)
Rabbit anti collagen 1 antibody, by Abcam (1 mentions)
Rabbit anti c3 antibody, by Abcam (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Cyanine3 (cy3), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin antibody, by Abcam (1 mentions)
4 protocols using rabbit anti neun antibody
1
Immunofluorescence Staining of Brain Sections
2
Multimodal Neuroinflammation Analysis
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Sections were boiled in citric acid buffer and were treated with 0.3% (v/v) Triton X-100 and 10% (v/v) goat serum, they were incubated overnight at 4 °C with primary antibodies (rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody, Wako, Japan; mouse anti-NLRP3 antibody, Thermo Fisher, USA; mouse purified anti-β-amyloid, 1-42 antibody, BioLegend, USA; mouse purified anti-β-amyloid, 1-40 antibody, BioLegend, USA; rabbit anti-collagen I antibody, Abcam, USA; rat anti-LAMP2 antibody, Abcam, USA; mouse anti-GFAP antibody, Sigma, USA; rabbit anti-C3 antibody, Abcam, USA; rabbit anti-NeuN antibody, Millipore, USA; mouse anti-cleaved caspase-3 antibody, Cell Signaling Technology, USA) and then incubated with secondary antibodies. Apoptosis was detected using a transferase-mediated deoxyuridine triphosphate-biotin nick end labeling Kit (TUNEL Apoptosis Detection Kit, Roche, Switzerland). Slices were embedded using Fluoroshield™ with DAPI (Sigma, USA). Images were acquired using a Nikon fluorescence microscope (Nikon, Japan) or a confocal microscope (Leica, Germany).
3
Immunodetection of Brain IL-1RA Penetration
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Immunohistochemical staining was also used to detect penetration of IL-1RA and IL-1RA-PEP in the brain. Coronal frozen sections (20 μm) were produced in the same manner as described above. The sections were washed with PBS. They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK). Brain sections were washed and then incubated with appropriate fluorochrome-conjugated secondary antibodies (Alexa Fluor 488 or Cy® 3, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The sections were counterstained with DAPI. Images were photographed with laser scanning confocal fluorescence microscopy (Zeiss LSM780, Carl Zeiss, Jena, Germany).
4
Neuronal Marker Expression Analysis
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NPCs in 60 mm dishes incubated for 3 days in differentiation media with or without aucubin were lysed in 80 µl of ice-cold radioimmunoprecipitation assay (RIPA) buffer (cat # R0278, Sigma-Aldrich) containing a protease inhibitor cocktail and cleared via 10 min of centrifugation at 13,000×g. Following determination of protein concentration using Bradford protein assay kits (Cat. No. B6916, Bio-Rad, Hercules, California, USA), 10~20 µg of proteins were separated on 10 % sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and probed for various neuronal markers and β-actin. The primary antibodies used were mouse anti-vGluT1 antibody (1:400, Chemicon), rabbit anti-GAD65/67 antibody (1:500, Cat. No. PA5-36080, Chemicon), rabbit anti-NeuN antibody (1:5000, Cat. No. ab104225, Abcam) and mouse anti-β-actin antibody (1:1000, Cat. No. sc-47778, Santa Cruz, Delaware, California, USA). Signals were visualized using enhanced chemiluminescence (LumiGLO, KPL Europe, Guildford, UK) as recommended by the manufacturer. The intensity of each band was quantified with a densitometric scanner using an image analysis program.
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