The expression levels of caspase-9 and GFAP in the hippocampal tissues of TLE rats (N = 5, in each group) were detected by ICH. Briefly, paraffin sections of hippocampal tissue were dewaxed and rehydrated with ethanol. Then, antigen retrieval was performed by incubation in 3% hydrogen peroxide for 10 min. Next, the sections were blocked with 5% bovine serum albumin (BSA) for 20 min, and incubated with primary antibodies (anti-caspase-9, 1:100, Epitomics, Burlingame, CA, USA; anti-GFAP, 1:100, Proteintech Group, Rosemont, IL, USA) overnight at 4 °C. After washing with PBS thrice, the sections were incubated with the secondary antibody (Wuhan Google, Wuhan, China) at 4 °C for 50 min. After staining with diaminobenzidine and haematoxylin, the tissue sections were observed under a microscope. The relative expression levels of caspase-9 and GFAP were quantitatively analysed with Image-Pro Plus 6.0 software.
Anti gfap
Manufactured by Proteintech
Sourced in United States
Anti-GFAP is a primary antibody used for the detection and quantification of Glial Fibrillary Acidic Protein (GFAP) in various samples. GFAP is a type III intermediate filament protein that is frequently used as a marker for astrocytes and other glial cells in the central nervous system.
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Lab products found in correlation
Anti iba1, by Proteintech (3 mentions)
Anti caspase 9, by Abcam (1 mentions)
Iba 1, by Proteintech (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Anti neun, by Cell Signaling Technology (1 mentions)
Nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti bdnf, by Abcam (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti sirt1, by Proteintech (1 mentions)
Anti tnf α, by Proteintech (1 mentions)
Anti pgc 1α, by Abcam (1 mentions)
Anti ng2, by Merck Group (1 mentions)
Anti psd95, by Cell Signaling Technology (1 mentions)
Anti synapsin 1, by Cell Signaling Technology (1 mentions)
Ah 2 light microscope, by Olympus (1 mentions)
Dab peroxidase substrate, by Beyotime (1 mentions)
Anti map2, by Proteintech (1 mentions)
Anti mbp, by Abcam (1 mentions)
Cy3 conjugated secondary antibody, by Proteintech (1 mentions)
9 protocols using anti gfap
1
Assessing Apoptosis and Astrogliosis in TLE Rat Hippocampus
2
Immunofluorescence Staining of Brain Tissue in Traumatic Brain Injury
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Mice in the normal control, TBI, TBI+CeO2, and TBI+Cr/CeO2 groups were perfused, and the brains were removed and fixed in 4% paraformaldehyde. Then, the brain tissue was made into paraffin slices. The slices were stained by the following procedures: dewaxing in xylene, dehydration in gradient ethanol (100%, 95%, 80%, and 70%), antigen retrieval in sodium citrate or EDTA solutions, addition of primary antibody overnight at 4 ℃, incubation with goat anti rabbit IgG 488 or 594 secondary antibody for 1 h, staining of nuclei with DAPI for 5 min and sealing with fluorescence quencher. For primary antibody, anti-GFAP, Iba1 and matrix metalloproteinase-9 (MMP-9) (Proteintech) were used to stain astrocytes, microglia and MMP-9, respectively. Notably, the staining process was carried out in the dark. Finally, the stained images were acquired by fluorescence microscope (EVOS, AMG) and immunofluorescence analysis was performed by Image J software.
3
Immunohistochemical Analysis of Mouse Brain
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Brain samples of mice were fixed in 4% paraformaldehyde, sequentially dehydrated in 20, 25, and 30% sucrose solution, frozen in OCT compound, and then prepared as 15-μm slices. Slices were incubated with indicated primary antibodies overnight at 4 °C, followed by incubation with appropriate secondary antibodies conjugated with fluorescence and DAPI for 60 min at room temperature. The fluorescence microscope images were acquired by an A1R (Nikon) confocal microscope. Antibodies used were: anti-NeuN (Cell Signaling Technology, 94403 S), anti-GFAP (Proteintech, 16825-1-AP), anti-Iba1 (Wako, 019–19741), and Alexa fluor 594-conjugated goat anti-rabbit IgG (H C L) secondary antibody (Thermo Fisher Scientific, A-11012).
4
Neuroinflammation and Cognitive Impairment
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The instruments and reagents used in these experiments were as follows: a 7.0T animal magnetic resonance instrument (Bruker, Germany); novel object recognition and test system (Boster Bioengineering, China); Y labyrinth video analysis system (Shanghai Xinruan Information Technology, China); high-fat feed (21% fat, 0.15% cholesterol, Jiangsu Medisen Biomedicine, China); isoflurane (Shenzhen Reward, China); immunohistochemistry kit and DAB staining kit (Boster Bioengineering, China); and haematoxylin staining solution and eosin staining solution (Beijing Solebao Technology, China). The primary antibodies in this study were as follows: anti-SIRT1 (Proteintech, USA), anti-TNF-α (Proteintech, USA), anti-β-actin (Proteintech, USA), anti-IBA1 (Proteintech, USA), anti-GFAP (Proteintech, USA), anti-PGC-1α (Abcam, USA), anti-BDNF (Abcam, USA), anti-IL-1β (Abcam, USA), NF-κBp65 antibody (Cell Signaling Technology, USA), goat anti-mouse/anti-rabbit secondary antibody (Proteintech, USA), and antibody diluent (Beijing Biyuntian, China).
5
Immunohistochemical Analysis of Myelination and Neuroinflammation
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Fixed brains were paraffin-embedded and cut into 4-μm sections. After antigen retrieval, the sections were blocked and stained with anti-Oligo2 (1:500, Proteintech), anti-MBP (1:200, Abcam), anti-NG2 (1:100, Millipore), anti-Iba1 (1:500, Proteintech), anti-GFAP (1:500, Proteintech), anti-MAP2 (1:500, Proteintech), anti-PSD95 (1:500, Cell Signaling Technology), or anti-Synapsin I (1:500, Cell Signaling Technology). Then, the sections were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1:500, Abbkine, China) or (HRP)-conjugated anti-mouse antibody (1:500, Abbkine, China). The sections were developed using DAB peroxidase substrate (Beyotime Biotechnology, China). The sections were photographed with an Olympus AH-2 light microscope (× 200; Olympus). Images were imported into ImageJ for quantification. Briefly, the well-stained area on the images was first selected; then, the mean integrated optical density value of the area was calculated by ImageJ. According to the mean integrated optical density of the control group, the relative density of the EAE group and the MCC950 treatment group was calculated.
6
Immunofluorescence Staining for Glioma Stem Cells
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Immunofluorescence staining was performed following a standard protocol. Briefly, adhered cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. Subsequently, the samples were permeabilized in 0.5% Triton X-100 and blocked in 5% blocking buffer for 1 h. The cells were then incubated at 4 °C overnight with specific primary antibody (anti-vimentin, anti-FZD7). After washing with PBS three times, the cells were incubated with fluorescence-conjugated secondary antibody (Proteintech) for 1 h and stained with DAPI (4′ 6-diamidino-2-phenylindole) for 10 min, and viewed under a fluorescence microscope.
For GSC identification, tumor spheres were placed on poly-L-ornithine (BD Biosciences)-coated glass coverslips, incubated with anti-CD133 (1:100, Proteintech), and stained with Cy3-conjugated secondary antibody (Proteintech). For the differentiation assay, tumor spheres were seeded in a 24-well plate and cultured in medium supplemented with 10% FBS for 5 days. Differentiated cells were incubated with anti-GFAP (glial fibrillary acidic protein) antibody (1:100, Proteintech), stained with Cy3-conjugated secondary antibody (Proteintech), and counterstained with DAPI.
For GSC identification, tumor spheres were placed on poly-L-ornithine (BD Biosciences)-coated glass coverslips, incubated with anti-CD133 (1:100, Proteintech), and stained with Cy3-conjugated secondary antibody (Proteintech). For the differentiation assay, tumor spheres were seeded in a 24-well plate and cultured in medium supplemented with 10% FBS for 5 days. Differentiated cells were incubated with anti-GFAP (glial fibrillary acidic protein) antibody (1:100, Proteintech), stained with Cy3-conjugated secondary antibody (Proteintech), and counterstained with DAPI.
7
Dual Immunofluorescence Labeling of MMP-9, Claudin-5, GFAP, and NeuN
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After standard histological procedures, sections were treated by high pressure antigen retrieval for 2 minutes and blocked with 5% goat serum at 37°C for 20 minutes. Afterwards, sections were incubated overnight at 4°C in mixtures of anti-MMP-9 antibody (rabbit, polyclonal, 1:50; Abcam)/anti-glial fibrillary acidic protein (GFAP) (mouse, monoclonal, 1:200; Proteintech), anti-claudin-5 antibody (rabbit, polyclonal, 1:100; Abcam)/anti-GFAP (mouse, monoclonal, 1:200; Proteintech), anti-MMP-9 antibody (rabbit, polyclonal, 1:50; Abcam)/anti-NeuN (mouse, monoclonal, 1:200; Abcam), anti-claudin-5 antibody (rabbit, polyclonal, 1:100; Abcam)/anti-NeuN (mouse, monoclonal, 1:200; Abcam), or anti-claudin-5 antibody (rabbit, polyclonal, 1:100; Abcam)/anti-MMP-9 (mouse, monoclonal, 1:50; Abcam). After washing in PBS, sections were incubated with fluorescence secondary antibody IgG (monoclonal, anti-mouse Alexa Fluor488-conjugated, green, 1:100; Proteintech)/IgG (polyclonal, anti-rabbit Alexa Fluor 594-conjugated, red, 1:100; Proteintech) for 2 hours at 37°C. After DAPI mounting, sections were imaged using a fluorescence microscope (Olympus).
8
GFAP and Iba-1 Immunohistochemistry in Brain Tissues
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The sections from paraffin-embedded brain were used to label target antibody, and the experiment was conducted as previously described (48 (link)). Sections were deparaffinized and pretreated for 10 min by an autoclave in Tris-EDTA buffer (pH 9.0) before undergoing blocking with 5% normal goat serum. The sections were washed in PBS and incubated with anti-GFAP (Proteintech) or anti-Iba-1 (Proteintech) overnight at 4°C. On the next day, the sections were washed with PBS, and then the slides were incubated with the appropriate secondary antibody for 1 h at room temperature. After being washed with PBS adequately, the slides were counterstained with hematoxylin and mounted with Aquatex. Pictures were captured using Olympus BX53 microscope and analyzed using ImagePro-Plus software.
9
Spinal Cord Injury Histological Analysis
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In vitro-cultured cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes. All mice were deeply anaesthetized and transcardially perfused with 4% PFA 10 weeks after injury. The dissected spinal cords were embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan) and sectioned in the axial plane at a thickness of 12 µm on a cryostat (Leica Biosystems, Wetzlar, Germany). The samples were stained with the following primary antibodies: anti-GFP (goat IgG, 1:500, Rockland, PA, USA), anti-mCherry (rabbit IgG, 1:400, Abcam, Cambridge, UK), anti-pan-ELAVL (mouse IgG1, 1:200, Sigma-Aldrich), anti-GFAP (rabbit IgG, 1:2000, Proteintech, IL, USA), anti-APC (mouse IgG2b, 1:300, Abcam), antihuman GFAP (mouse IgG1, 1:2000, Takara Bio, Shiga, Japan), anti-CNPase (mouse IgG1, 1:2000, Sigma-Aldrich), anti-Ki-67 (rabbit IgG, 1:2000, Leica Biosystems), anti-Nestin (rabbit IgG, 1;200, IBL, Gunma, Japan), anti-HNA (msIgG1, 1:100, Millipore, Darmstadt, Germany), anti-Fos (rabbit IgG, 1:400, Abcam), and STEM121 (msIgG1, 1:200, Takara Bio). The nuclei were stained with Hoechst 33258 (10 µg/ml, Sigma-Aldrich). All images were obtained using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan) or confocal laser scanning microscope (LSM 780; Carl Zeiss, Jena, Germany).
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