The hippocampus was separated from the brain tissue and homogenized and lysed by adding a protein extraction solution (PRO-PREP, iNtRON, Sungnam, South Korea). After measuring the total protein concentration using Bradford reagent (Bio-Rad, Hercules, CA, USA), western blotting was performed as previously described (Han et al., 2019 (link)). Protein-specific primary antibodies were purchased from Novus Biologicals (anti-APP; Littleton, CO, USA), Santa Cruz Biotechnology (anti-GFAP and anti-β-actin; Dallas, TX, USA), Cell Signaling Technology (anti-iNOS, anti-COX-2, anti-p65, and anti-p50; Danvers, MA, USA), and Abcam [anti-BACE1, anti-ionized calcium-binding adapter molecule 1 (IBA-1), and anti-Foxp3; Cambridge, MA, USA]. Secondary antibodies were purchased from Santa Cruz Biotechnology (anti-mouse, anti-rabbit, and anti-goat).
Anti gfap
Manufactured by Cell Signaling Technology
Sourced in United States, Japan, Germany
Anti-GFAP is a primary antibody that recognizes the glial fibrillary acidic protein (GFAP), a cytoskeletal protein found in astrocytes and other glial cells. This antibody can be used to identify and visualize astrocytes in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti iba1, by Fujifilm (9 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Anti neun, by Merck Group (6 mentions)
Anti β actin, by Cell Signaling Technology (5 mentions)
Anti iba1, by Cell Signaling Technology (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Anti gfp, by Thermo Fisher Scientific (4 mentions)
Anti phospho akt, by Cell Signaling Technology (3 mentions)
Anti erk, by Cell Signaling Technology (3 mentions)
Anti neun, by Cell Signaling Technology (3 mentions)
Anti β actin, by Bioworld Technology (3 mentions)
Anti cd11b, by Bio-Rad (3 mentions)
Anti neun, by Abcam (3 mentions)
Alexa fluor 488, by Cell Signaling Technology (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Alexa fluor 594 secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti cc1, by Merck Group (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Anti phospho erk, by Cell Signaling Technology (2 mentions)
Anti trpv1, by Novus Biologicals (2 mentions)
69 protocols using anti gfap
1
Hippocampal Protein Expression Analysis
2
Histological Analysis of Hippocampal Neurons
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Hippocampal tissues were dehydrated and embedded in paraffin after fixation as described previously for hematoxylin‐eosin (HE) staining and immunohistochemistry.
45 (link) The paraffin‐embedded tissues were sectioned at a thickness of 5 μm. Some of the sections were stained with HE according to the standard protocol, and the morphology and structure of neurons in the CA1 region of the hippocampus were observed under a light microscope. Some sections were incubated with rabbit mAb anti‐GFAP (#80788, Cell Signaling Technology) and anti‐IBA1 (ionized calcium binding adaptor molecule 1; #17198, Cell Signaling Technology) and then incubated with Alexa Fluor 488 (#4412, Cell Signaling Technology) and 594 (#8889, Cell Signaling Technology) conjugated with goat anti‐rabbit IgG at room temperature for 2 h. The photomicrographs of the sections were captured by laser scanning confocal microscopy (Fluo‐View‐FV1000, Olympus, Japan), astrocytes and microglia morphologies were categorized with ImageJ protocols.
46
45 (link) The paraffin‐embedded tissues were sectioned at a thickness of 5 μm. Some of the sections were stained with HE according to the standard protocol, and the morphology and structure of neurons in the CA1 region of the hippocampus were observed under a light microscope. Some sections were incubated with rabbit mAb anti‐GFAP (#80788, Cell Signaling Technology) and anti‐IBA1 (ionized calcium binding adaptor molecule 1; #17198, Cell Signaling Technology) and then incubated with Alexa Fluor 488 (#4412, Cell Signaling Technology) and 594 (#8889, Cell Signaling Technology) conjugated with goat anti‐rabbit IgG at room temperature for 2 h. The photomicrographs of the sections were captured by laser scanning confocal microscopy (Fluo‐View‐FV1000, Olympus, Japan), astrocytes and microglia morphologies were categorized with ImageJ protocols.
46
3
Quantifying Proliferating Cells in Ischemic Brain
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To analyze the proliferating cells, 18-μm thick brain sections were prepared using a cryostat (CM1950, Leica) from the animals of the second set. Sections were then blocked in 10% goat serum (#G9023, Sigma) in PBS solution and incubated with monoclonal rat anti-BrdU antibody (#ab6326, Abcam). To identify the BrdU(+) proliferating cells, double staining was performed using anti-NeuN (#MAB377C3, Millipore), anti-GFAP (#3656, Cell Signaling) or anti-Iba1 (#019-1974, Wako) antibodies overnight at 4°C. On the next day, sections were washed with PBS and incubated with the appropriate secondary antibodies for 1 h at room temperature. Sections were then imaged with a laser scanning confocal microscopy (LSM 780, Carl Zeiss). Nine different ROI in the ischemic striatum, each measuring 62,500 μm2, were evaluated. Numbers of double immunopositive cells were counted blindly.
4
Fluoromyelin and Immunostaining of Brain Sections
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Animals were perfused with 4% paraformaldehyde, tissue was embedded in optimal cutting medium, and 40 μm frozen sections were prepared. For fluoromyelin staining, slides were rehydrated in PBS for 20 min and then flooded with 1:300 diluted stain for 20 min. Counterstaining was done using Hoechst stain for 5 min, and slides were mounted using vectashield mounting medium (Vector Laboratories). Slides were imaged using the Zeiss LSM 700. For antibody staining, sections were incubated with primary anti-RelB, anti-GFAP, anti-p65, anti-phospho-p65 (all 1:300, Cell Signaling), anti-CC1 (1:200, Millipore), or anti-Iba1 (1:500, Wako) antibodies overnight at 4 °C. Subsequently, sections were incubated with Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (1:500, Invitrogen) for 1 h at room temperature. Slides were mounted and imaged as described above. No fluorescence crossover was found between the channels, and images were collected separately using the appropriate laser excitation. Images were analyzed using ImageJ.
5
Antibody Sources for Protein Analysis
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DT and TMZ were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The proteasome inhibitor MG132 was purchased from Calbiochem (San Diego, CA, USA). Monoclonal antibodies were purchased from the following companies: anti-CA9, anti-PARP-1, and anti-uPA antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-HIF-1α and anti-HIF-1β antibodies from BD Biosciences (San Jose, CA, USA); anti-phospho-JNK and anti-JNK antibodies from Promega (Madison, WI, USA); anti-actin antibody from ICN (Costa Mesa, CA, USA); anti-CD133 and anti-VEGF antibodies from Abcam (USA); anti-β-Tubulin (Tuj1) antibody from Covance (USA); anti-Nestin antibody from Chemicon (USA); anti-SOX-2 antibody from R&D Systems (Minneapolis, MN, USA); and anti-Bmi1, anti-Musashi, anti-GFAP, anti-phospho-ERK, anti-ERK, anti-phospho-p38, anti-p38, anti-MMP-2, anti-MMP-9, and anti-phospho-Akt antibodies from Cell Signaling (Beverly, MA, USA).
6
Immunophenotyping of BEC Cell Line
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Immunohistochemical analysis was performed to confirm the phenotype of the human BEC cell line. Cells were rinsed twice with cold PBS, fixed with ice-cold Methanol for 10 min, and then permeabilized with 0.2% Triton X-100 (5 min; room temperature). Cells were then blocked with 0.1% BSA in PBS (1 h), and incubated with primary antibodies anti-P-gp (Santa Cruz Biotechnology, Dallas, Texas, USA, #SC-55510, 1:200), anti-BCRP (Millipore, #MAB146, 1:200), anti-Claudin-5 (Invitrogen #352588, 1:200), anti-PECAM-1 (Dako, Burloak, ON, Canada, #M0823, 1:200) and anti-GFAP (Cell Signaling Technology, Whitby, ON, Canada, #3670, 1:300) overnight at 4°C. The primary antibodies anti-rabbit IgG (Abcam, Toronto, ON, Canada, #ab171870), anti-mouse IgG1 (Dako, #X0931), anti-mouse IgG2a (Dako, #X0943), and anti-mouse IgG2b (Dako, #X0944) were used as isotype controls. Subsequently, cells were washed (3X) and incubated with fluorochrome-labeled secondary antibody Alexa Fluor 488 (Molecular Probes Inc., Eugene, OR, USA, 1:300) and counterstained with DAPI (1 mg/mL, 60 min). The area and intensity of antibody staining were analyzed, and representative images were obtained using a spinning disc confocal fluorescent microscope (Leica DMI6000 B, Concord, ON, Canada) at 40X magnification.
7
Immunofluorescence Characterization of HIBD and OGD Astrocyte Responses
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Immunofluorescence staining was carried out to detect GFAP expression in mice after HIBD as well as TRPV1, GFAP, and iNOS expression in astrocytes after OGD. Brain serial coronal sections and astrocytes were washed with PBS before fixed with 4% paraformaldehyde at room temperature for 30 min. Subsequently, they were incubated with a blocking solution (5% FBS) for 30 min at 37 °C. Then, they were incubated with the anti-TRPV1 (Novus Biologicals, #9886, 1:300), anti-GFAP (Cell Signaling, #3670, 1:200), or anti-iNOS (Cell Signaling, #2985, 1:200) antibodies overnight at 4 °C. On the following day, they were washed and incubated with secondary antibodies Cy3-conjugated anti-IgG (Protein tech, SA00009-4, 1:30), Alexa Fluor® 488 Conjugates (Cell Signaling Technology, #4408, 1:200) for 1 h at 37 °C and DAPI (Beyotime, #C1002,1:2000) for 1 min at room temperature. Phalloidin staining was performed following the same protocol used for immunofluorescence (IF) while astrocytes were incubated with FITC-phalloidin (sigma, #p5282, 5 μg/ml) for 1 h and DAPI for 1 min at 37 °C in the dark. Images were obtained using a confocal microscope (Leica-LCS-SP8-STED).
8
Immunocytochemistry of Neural Stem Cells
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The antibodies used in this study were anti-Ki67 (rabbit polyclonal 1:500; Abcam, Cambridge, MA, USA), anti-Nestin (mouse monoclonal 1:350; BD Biosciences, San Jose, CA, USA), anti-Sox2 (rabbit polyclonal 1:500, Abcam), anti-Tuj1 (mouse polyclonal 1:200; Covance, Princeton, NJ, USA), anti-MAP2 (rabbit polyclonal 1:200; Chemicon, Temecula, CA, USA), and anti-GFAP (rabbit 1:500; Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies were anti-rabbit Alexa Fluor 488-conjugated, anti-mouse Alexa Fluor 488-conjugated, anti-rabbit Alexa Fluor 555-conjugated, and anti-mouse Alexa Fluor 555-conjugated immunoglobulin G (1:200 dilution; Molecular Probes, Eugene, OR, USA). The protease inhibitor cocktail was from Roche Applied Science (Indianapolis, IN, USA).
9
Immunoblot Analysis of Protein Targets
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Immunoblot analysis was performed as previously described (44 (link)). Antibodies were used as follows: anti-USP25 (Abcam, Cambridge, MA, USA; ab187156, 1:1000), anti-APP (Millipore, Billerica, MA, USA; MAB348, 1:1000), anti-Iba1 (Wako Pure Chemical, Osaka, Japan; 016-20001, 1:500), anti-GFAP (glial fibrillary acidic protein) (Cell Signaling Technology, Danvers, MA, USA; 3670, 1:1000), anti–β-III-tubulin (Abcam, ab18207, 1:1000), anti-HA (hemagglutinin) (Sigma-Aldrich, St. Louis, MO, USA; H6908, 1:1000), anti-ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-8017, 1:500), anti–β-actin (Xmbcss, Xiamen, China; bc001, 1:2000), and horseradish peroxidase (HRP)–conjugated secondary antibodies (Thermo Fisher Scientific, 31430 or 31460; 1:3000).
10
Immunoblotting Protocol for Protein Quantification
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