The brain tissue was fixed with 4% paraformaldehyde at 4°C, after the brain tissues sink to the bottom of the bottle and after dehydrating by concentration of gradient (10%, 20%, 30%, and 5%), and then, 12 μm frozen sections were prepared and examined. All sections were blocked with blocking solution (10% goat serum, 3% BSA, and 0.1% Triton X-100) for 1 h at 37°C, incubating overnight with antibody WISP1 (diluted 1 : 500; Santa Cruz), anti-GFAP (1 : 400; BD Pharmingen), anti-NeuN (1 : 1000; Abcam), β-catenin (1 : 500; Santa Cruz), anti-cleaved caspase-3 (1 : 400; Cell Signaling Technology), anti-LC3 (1 : 500; Cell Signaling Technology), and anti-Iba1 (1 : 1000; Wako) and then washing them with 0.01 M PBS for 10 min at 3 times and followed by incubating with a mixture of FITC- or Cy3-conjugated secondary antibodies for 2 h at room temperature and then being washed again in PBS for 10 min at 3 times. The stained sections were examined with a Leica fluorescence microscope (Leica DM 5000B, Germany).
Anti gfap
Manufactured by BD
Sourced in United States
Anti-GFAP is a laboratory reagent used to detect the presence of the Glial Fibrillary Acidic Protein (GFAP) in biological samples. GFAP is a cytoskeletal protein found primarily in astrocytes, a type of glial cell in the central nervous system. Anti-GFAP can be used in various analytical techniques, such as immunohistochemistry and Western blotting, to identify and quantify GFAP levels.
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Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti sox2, by Cell Signaling Technology (2 mentions)
Anti olig2, by Merck Group (2 mentions)
Anti β actin peroxidase conjugated antibody, by Merck Group (2 mentions)
Benzonase nuclease, by Merck Group (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Supersignal substrate, by Thermo Fisher Scientific (2 mentions)
Anti snap25, by Abcam (2 mentions)
Anti snap23, by Abcam (2 mentions)
Anti β tubulin, by Merck Group (2 mentions)
Anti glur1, by Merck Group (2 mentions)
Anti nr1, by Merck Group (2 mentions)
Anti nr2b, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Anti ki67, by Abcam (2 mentions)
Anti iba1, by Fujifilm (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Wisp 1, by Santa Cruz Biotechnology (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
13 protocols using anti gfap
1
Immunofluorescent Staining of Brain Tissue
2
Molecular Mechanisms of Glioblastoma Response to Temozolomide
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Reagent and antibody sources were as follows: AG1478 (Calbiochem/Merck, Darmstadt, Germany), BMP4 (R&D Systems, Minneapolis, MN, USA), DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), temozolomide (TMZ) and anti-β-Actin-peroxidase conjugated antibody (Sigma-Aldrich, Munich, Germany), anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), anti-BIM, anti-cleaved caspase 3, anti-cleaved caspase 7, anti-cleaved PARP (poly (ADP-ribose) polymerase-1), anti-EGF Receptor, anti-phospho-EGF receptor (Tyr1068), anti-FOXO3a, anti-phospho-FOXO3a (Thr32), anti-phospho-FOXO3a (Ser253), anti-phospho-FOXO3a (Ser318/321), anti-phospho-Rb (Ser807/811), anti-SMAD1, anti-SMAD3, anti-SMAD4, anti-SMAD5, anti-phospho-SMAD1/5 (Ser463/465), anti-phospho-SMAD3 (Ser423/425), anti-p27Kip1, anti-SOX2 (Cell Signaling Technology, Beverly MA, USA), anti-OLIG2, anti-β-Tubulin beta III isoform (Millipore, Temecula, CA, USA), anti-CYCLIN B1, p21CIP1 (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-GFAP (BD Pharmingen San Jose, CA), anti-NESTIN (R&D Systems, Minneapolis, MN, USA), and anti-CYCLIN D1 (ThermoFisher Scientific, Waltham, MA USA).
3
Immunofluorescence Staining of Microchip Cultures
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Cultures in the microchips were washed once with DPBS and fixed in 4% paraformaldehyde (Fisher Scientific) for 20 min at room temperature. The chips were then washed once with DPBS, blocked/permeabilized in saponin buffer (0.1% saponin, 1% BSA, 10% FBS in DPBS) for 1 h at room temperature, and incubated overnight at 4 °C with primary antibodies (1:50 dilution) in saponin buffer without FBS. The following antibodies were used: anti-GFAP (BD Biosciences), anti-Alpha-Smooth Muscle Actin (aSMA) (Sigma), anti-Glut1 (Novus Biologicals, Toronto, Canada), anti-ZO-1 (Invitrogen). The next day, the cells were washed twice with DPBS, then incubated in Alexa-conjugated secondary antibodies of the appropriate species (goat-anti rabbit, goat-anti mouse, donkey anti-mouse; Alexa Fluor 488; Alexa Fluor 555; Thermo Fisher Scientific) and DAPI nuclear stain diluted 1:500 in saponin buffer without FBS for 1 h at room temperature. Lastly, the cells were washed three times with DPBS and stored at 4 °C.
4
Hippocampal Protein Expression Analysis in Injured Mice
5
Immunostaining of Neuronal and Glial Cells
6
Immunofluorescent Characterization of Cell Cultures
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Cell monolayers or tissue patches were fixed in 2% paraformaldehyde (15 min), permeabilized in 0.5% Triton-X (30 min) and blocked in a 5:1 solution of 1% BSA and chicken serum (30 min). The following primary antibodies (1 h incubation) were used: anti-sarcomeric α-actinin (Sigma, a7811, 1:200), anti-Cx43 (Life Technologies, 71-0700, 1:100), anti-vimentin (Sigma, v6630, 1:500), anti-smooth muscle actin (Sigma, a2547, 1:200), anti-GFAP (BD Biosciences, 561483, 1:100) and anti-Ki67 (Abcam, ab15580, 1:200). Secondary antibodies (1 h incubation) included the following: Alexa488 (Life Technologies, A-21200 or A-21441, 1:200), Alexa594 (Life Technologies, A-21201 or A-21442, 1:200), Alexa647 (Life Technologies, A-21463, 1:200), Alexa488-conjugated phalloidin (Life Technologies, A12379, 1:300) and 4,6-diamidino-2-phenylindole (Sigma, 1:300). All immunostaining steps were performed at room temperature. Fluorescence images were acquired using inverted fluorescence (Nikon TE2000) or confocal (Leica SP5) microscope and processed with ImageJ software.
7
Autophagy Regulation in Stem Cells
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Reagent and antibody sources were as follows: acridine orange, ATRA (all-trans-retinoic acid), bafilomycin A1 (BafA1), BIX01294 trihydrochloride hydrate, DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), laminin, 3-methyladenine (3MA), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), anti-LC3 (catalog no. L7543) and anti-β-Actin-peroxidase conjugated antibody (catalog no. A3854) (Sigma-Aldrich, Munich, Germany), anti-ATG5 (catalog no. 2630), anti-ATG7 (catalog no. 2631), anti-cleaved caspase 3 (catalog no. 9661), anti-cleaved caspase 7 (catalog no. 9491), anti-cleaved PARP (poly (ADP-ribose) polymerase-1) (catalog no. 9541), anti-SOX2 (catalog no. 3579) (Cell Signaling Technology, Beverly MA, USA), anti-ULK1 (catalog no. sc-33182) (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-Beclin1 (catalog no. 612112), anti-GFAP (catalog no. 556330) (BD Pharmingen San Jose, CA, USA), anti-H3K4me3 (catalog no. 07-473), anti-H3K27me3 (catalog no. ABE44), anti-OLIG2 (catalog no. AB9610), anti-Tubulin beta III isoform (catalog no. MAB1637) (Millipore, Temecula, CA, USA), anti-H3K9me2 (catalog no. ab1220), anti-G9a (catalog no. ab40542) (Abcam, Cambridge, UK), anti-RNA Pol II (catalog no. 39097) (Active Motif, Carlsbad, CA, USA) and anti-NESTIN (catalog no. MAB1259) (R&D Systems, Minneapolis, MN, USA).
8
Hippocampal Protein Expression Analysis in Injured Mice
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Ipsilateral hippocampal tissues from sham and CCI injured mice were homogenized in RIPA buffer with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and benzonase nuclease (Millipore Corporation, Billerica, MA) and mixed by rocking at 4 °C for at least 15 min. Tissues were centrifuged, supernatant was recovered, samples were diluted and standardized to protein concentrations. Protein samples were separated on 8–10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were then blocked with 5% milk or 5% BSA in 0.1 M phosphate buffer with 0.1% Tween-20 for 1 h at room temperature (RT) and incubated overnight at 4 °C with primary antibodies. Membranes were incubated for 1 h at RT with HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). Bands were visualized using SuperSignal substrate (ThermoScientific, Pittsburg, PA). The following primary antibodies were used: anti-GluR1, anti-NR1, anti-NR2B (EMD Millipore, Billerica, MA), anti-GFAP (BD Biosciences, San Jose, CA), anti-SNAP25, anti-SNAP23 (ABCAM, Cambridge, MA) and anti-β-tubulin (Sigma-Aldrich, St. Louis, MO) antibodies. ImageJ was used to perform density analysis. Protein measurements were standardized to β-tubulin and normalized to average WT sham signals.
9
Quantifying Hippocampal Cell Proliferation by FACS
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Fluorescence-activated cell sorting (FACS) acquisition and analyses were performed as previously described [34 (link)]. Briefly, sample preparation was performed by using a APC BrdU Flow kit (Cat# 552598, BD Pharmingen™) according to the manufacturer’s instructions. Rat hippocampal tissues were fixed in 4% PFA for 30 min, followed by mincing on ice and incubation in 4% PFA for 10 min. Tissues were then washed in PBS-T before enzymatic digestion by StemPro™ Accutase™ Cell Dissociation Reagent (Cat# A1110501, Thermo Fisher). Cells were collected, fixed, and permeabilized with BD Cytofix/Cytoperm Buffer. Incorporated BrdU was revealed by incubation with DNase for 1 h at 37°C. Cells were washed with BD Perm/Wash Buffer and stained with fluorochrome-conjugated anti-BrdU (Cat# 552598, APC, BD Pharmingen), anti-NeuN (Cat# MAB377x, Alexa Fluor 488, Millipore), and anti-GFAP (Cat# 561483, PE, BD Pharmingen) antibodies. Single antibody-stained samples were first analyzed to optimize the laser intensity and the necessary compensation to eliminate fluorescence leakage and background signals before the all-stained samples were screened. Analysis of the 20000 DAPI+ events was performed on a NovoCyte Quanteon Flow Cytometer using NovoExpress software.
10
Immunocytochemistry of Neuronal and Glial Cells
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