The 10-μm thick coronal sections were incubated with 3% H2O2 for 5 min to inhibit endogenous peroxidase activity and then rinsed in PBS for 5 min, followed by incubation with primary anti-glial fibrillary acidic protein (GFAP) antibody (Abcam, USA) diluted by 1:200 (glial fibrillary acidic protein) at 4°C overnight. Following incubation with species-specific biotinylated secondary antibody, the sections were incubated with peroxidase-labeled streptavidin, and the color was developed with diaminobenzene (Brusco et al., 1997 (link)). Finally, the sections were counterstained with hematoxylin. Negative controls were processed simultaneously by omitting the primary antibodies.
Gfap antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The GFAP antibody is a laboratory reagent used to detect the presence of the Glial Fibrillary Acidic Protein (GFAP) in biological samples. GFAP is an intermediate filament protein that is primarily expressed in astrocytes, a type of glial cell in the central nervous system. This antibody can be used in various applications, such as immunohistochemistry, Western blotting, and flow cytometry, to visualize and quantify GFAP levels in cells and tissues.
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Lab products found in correlation
Neuronal nuclei (neun), by Abcam (2 mentions)
Diaminobenzidine dab, by Merck Group (1 mentions)
Rabbit polyclonalanti doublecortin dcx antibody, by Abcam (1 mentions)
Mouse monoclonal anti nestin antibody, by Abcam (1 mentions)
Lsm image examiner, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Dmire2, by Leica (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Prolong antifade, by Thermo Fisher Scientific (1 mentions)
Fluorochrome conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Dm2500, by Leica (1 mentions)
K5007, by Agilent Technologies (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Fluorescence microscopy, by Leica (1 mentions)
Iba1 antibody, by Abcam (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Anti bax antibody, by Abcam (1 mentions)
Iba 1, by Abcam (1 mentions)
Anti p erk5 antibody, by Santa Cruz Biotechnology (1 mentions)
29 protocols using gfap antibody
1
Immunohistochemical Analysis of GFAP
2
Immunohistochemical Analysis of Astrocyte Reactivity
3
Immunofluorescence Analysis of Neural Stem Cells
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The stored frozen sections were rinsed in 0.1 M PBS (3 times for 5 min each time) at
first, permeabilized with 0.5% Triton X-100 (Beyotime, Shanghai, China) and incubated with
3% bovine serum albumin (BSA, Boster, Wuhan, China) at room temperature. The sections were
incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal
anti-Ki-67 antibody (1:200, Abcam, Cambridge, MA, USA), rabbit polyclonal
anti-doublecortin (DCX) antibody (1:100, Abcam), rabbit polyclonal anti-glial fibrillary
acidic protein (GFAP) antibody (1:500, Abcam), and mouse monoclonal anti-Nestin antibody
(1:100, Abcam). Afterward, the sections were incubated in the corresponding secondary
antibodies, goat anti-rabbit IgG (Alexa Fluor 555, Invitrogen, Carlsbad, CA, USA) or goat
anti-mouse IgG (Alexa Fluor 488, Invitrogen), at 37°C for 1 h, and
4′,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei at room temperature for 10
min. Slides were made for observation and storage. Immunofluorescence was examined by
confocal microscopy (LSM800, ZEISS, Oberkochen, Germany), and images were obtained using
an LSM Image Examiner. The numbers of Nestin-, DCX-, GFAP-, and Ki-67-positive cells were
counted using ImageJ (National Institutes of Health, Bethesda, MD, USA).
first, permeabilized with 0.5% Triton X-100 (Beyotime, Shanghai, China) and incubated with
3% bovine serum albumin (BSA, Boster, Wuhan, China) at room temperature. The sections were
incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal
anti-Ki-67 antibody (1:200, Abcam, Cambridge, MA, USA), rabbit polyclonal
anti-doublecortin (DCX) antibody (1:100, Abcam), rabbit polyclonal anti-glial fibrillary
acidic protein (GFAP) antibody (1:500, Abcam), and mouse monoclonal anti-Nestin antibody
(1:100, Abcam). Afterward, the sections were incubated in the corresponding secondary
antibodies, goat anti-rabbit IgG (Alexa Fluor 555, Invitrogen, Carlsbad, CA, USA) or goat
anti-mouse IgG (Alexa Fluor 488, Invitrogen), at 37°C for 1 h, and
4′,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei at room temperature for 10
min. Slides were made for observation and storage. Immunofluorescence was examined by
confocal microscopy (LSM800, ZEISS, Oberkochen, Germany), and images were obtained using
an LSM Image Examiner. The numbers of Nestin-, DCX-, GFAP-, and Ki-67-positive cells were
counted using ImageJ (National Institutes of Health, Bethesda, MD, USA).
4
Immunohistochemical Analysis of SCI
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Specific proteins were identified using immunohistochemical staining as described in our previous study. Briefly, the samples of SCI were dissected and post-fixed in 4% paraformaldehyde over 24 h. After being blocked by 5% goat serum, the coronal sections (4 μm) were stained with the anti-glial fibrillary acidic protein (GFAP) antibody (1:5,000 dilution; Abcam) and the anti-neurofilament-H antibody (1:200 dilution; CST). After incubation with the primary antibody at 4°C for 12 h, the sections were incubated with the fluorescent secondary antibody kits (Cy3 Goat anti-Rabbit and FITC Goat anti-Mouse) according to the manufacturer’s instructions. Nuclei were counterstained for 3 min using DAPI. All samples were examined with a fluorescence microscope (Leica DM IRE2; Leica Microsystems, Wetzlar, Germany). The heart, liver, spleen, lung and kidneys were also excised and stained with hematoxylin–eosin (H&E). Quantification of neurofilament and GFAP was performed by counting the number of positive cells in 15 high-power visual fields randomly. More than five sections of each sample were considered.
5
Astrocyte Protein Cleavage Assay
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Rat primary astrocytes were cultured on CC2 glass chamber slides (Nunc). Astrocytes were rinsed 2x with DMEM, and fixed with 4% paraformaldehyde for 5 minutes. Cells were washed with 50 mM Tris pH 7.4, 150 mM NaCl (TBS), then permeabilized with 0.1% Triton X-100 for 5 minutes. After washing, cells were digested with 1 μg/ml rat calpain-2 in TBS, 10 mM CaCl2, 20 mM DTT for 30 min. Undigested controls were treated in parallel with TBS, 10 mM EGTA, 20 mM DTT (no enzyme). Cells were washed with TBS, 10 mM EGTA, and post-fixed with 4% paraformaldehyde for 5 minutes. Cells were blocked with 2% goat serum in PBS for 30 minutes. GFAP antibody (Abcam), human normal serum, or TBI sera were diluted in blocking solution, added to cells, and incubated overnight at 4°C. The next day, cells were washed 3 times with PBS. Fluorochrome-conjugated secondary antibodies (Jackson ImmunoResearch) and Hoechst (10 μg/ml; Life Technologies), diluted in blocking solution, were added to cells (30 minutes). Cells were washed with PBS, mounted using ProLong Antifade (Life Technologies), and imaged with an Olympus 1X81-DSU spinning disk confocal microscope.
6
Quantifying Amyloid-Beta Plaques in Mouse Brain
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Three consecutive mice brains per each mouse were extracted for each mouse hemisphere fixed with 4% paraformaldehyde for 24 hours and immersed in 30% sucrose for 48 hours before cryosection. The other hemisphere was dissected to obtain cortex and hippocampus lysates. Fixed brain tissues were sliced with 35-μm thickness and detected the mice brains for reactive astrocytes by GFAP antibody (abcam, USA) followed by staining with 500 μM of ThS (Sigma-Aldrich, USA) dissolved in 50% ethanol for seven minutes in the dark for Aβ plaque visualization. The brain tissues were then destained with 100%, 90%, and 70% ethanol solutions for one minute each and washed twice in PBS for one minute each. Tissue images were obtained by a fluorescence microscope (Leica DM2500) with a LAS X software program. The number and area of Aβ plaques in cortex and hippocampus was analyzed by Image-J software.
7
Immunohistochemical Staining Protocol
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Monoclonal VEGFR-2- specific antibody (Cell signaling, Danvers, MA, USA) and GFAP antibody (Abcam, ab7260, Cambridge, UK) were used for immunohistochemical staining. Immunohistochemical labeling was scored incorporating both the intensity and the distribution of specific staining. H score was derived according to the modification of the previously reported method [20]. It was performed by a single investigator (UU) blinded to the study groups.
8
Immunohistochemical Analysis of Retinal GFAP
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The eyeballs were immersed in Formaldehyde, Aceticacid, Alcohol, Saline (FAS; Wuhan Servicebio Technology Co., Ltd., Wuhan, China), dehydrated in alcohol, and then embedded in paraffin. Paraffin-embedded tissue blocks were cut into serial 4 μm sections. Paraffin-embedded retina sections were dewaxed, rehydrated, washed with phosphate-buffered saline (PBS), incubated with 3% H2O2 to block endogenous peroxidase activity, and blocked with 3% bovine serum albumin. They were then incubated with GFAP antibody (Abcam, Cambridge, United Kingdom) at a dilution of 1:1,500 overnight at 4°C. The next day, the sections were washed with PBS three times for 5 min each and incubated with an HRP-conjugated secondary antibody (K5007, DAKO, Glostrup, Denmark) for 50 min at room temperature. After washing with PBS, the sections were stained with 3,3-diaminobenzidine tetrahydrochloride for approximately 1 min, counterstained in hematoxylin, dehydrated in absolute alcohol, cleared in xylene, and finally mounted in synthetic resin for microscopic examination.
9
Immunohistochemical Analysis of Alzheimer's Disease
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Free-floating sections were rinsed and blocked with 10% (w/v) normal donkey serum in Tris-buffered saline (TBS) for 1 h at room temperature. Then, the sections were incubated with primary antibodies for 48 h at 4°C. The following primary antibodies were used: mouse anti-Aβ monoclonal antibody (6E10; Covance, 1:500); mouse anti-phospho-tau (ser202, Thr205; AT-8; Thermo Fisher, 1:200); rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody (Abcam, 1:200); and rabbit anti-glial fibrillary acidic protein (GFAP) antibody (Abcam, 1:200). The sections were then rinsed with TBS and incubated for 2 h at room temperature with the respective secondary antibodies, Alexa Fluor-555-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 1:200), Alexa Fluor-488-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 1:200) or Alexa Fluor-488-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, 1:200). After a final rinse in TBS, the sections were mounted on chrome-alum gelatin-coated slides, air-dried and covered with glycerol (diluted in PBS, 1:1, v/v). Staining was visualized using fluorescence microscopy (Leica, Japan; n = 6 per group).
10
Immunohistochemical Analysis of Spinal Cord Injury
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At 24 h after SCI or surgery, tissues were removed and fixed in 10% (w/v) PBS-buffered formaldehyde, and 5-mm sections were prepared from paraffin-embedded tissues. Sections were incubated overnight with anti-p-ERK5 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Fas-ligand (FasL) antibody (1:500; Abcam, Cambridge, UK), anti-nuclear factor-κB (NF-κB) p65 S536 antibody (1:1000; Abcam), anti-Bax antibody (1:50; Abcam), or anti-Bcl-2 antibody (1:50; Abcam). Sections were washed with PBS, and then incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit immunoglobulin G (IgG) and avidin-biotin-peroxidase complex. For double immunofluorescence, spinal tissues were incubated with a mixture of anti-p-ERK5 antibody and anti-NeuN antibody (1:500; Abcam), or anti-ionized calcium binding adapter molecule 1 (Iba1) (1:500; Abcam) antibody, or anti-glial fibrillary acidic protein (GFAP) antibody (1:500; Abcam) overnight at 4°C. The stained sections were examined under a fluorescence microscope, and images were captured with a charge-coupled device spot camera.
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