Mice were perfused with 4% paraformaldehyde in PBS. Brains were rapidly removed and post-fixed for 4 h in the same fixative, and placed in 20% sucrose in PBS at 4 °C until they sank. Sections were coronally cut at 35 µm thickness on a sliding microtome. Four sets of serial sections were collected in glass vials. After blocking at room temperature for 1 hour in NGS (10% normal goat serum, 0.2% Triton X-100, and 0.02% NaN3 in TBS), free-floating sections were incubated ON at 4°C with anti-GFAP monoclonal antibody (Sigma-Aldrich, dilution 1:1000) in blocking solution. AlexaFluor 488 secondary antibody (Invitrogen, dilution 1:500) was used for 2 hours at RT. Sections were washed three times for 10 min with PBS after incubation with each antibody. The sections were then mounted in ProLong Gold Anti-Fade Reagent with DAPI (Invitrogen), and examined by fluorescence microscopy. Nissl staining and image quantification were performed as previously described [33 (link)].
Anti gfap monoclonal antibody
Manufactured by Merck Group
Sourced in United States
The Anti-GFAP monoclonal antibody is a laboratory research tool designed to detect the presence of the Glial Fibrillary Acidic Protein (GFAP) in biological samples. GFAP is a commonly used marker for astrocytes, a type of glial cell in the central nervous system. This antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to study the expression and distribution of GFAP in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Iba 1 polyclonal antibody, by Fujifilm (2 mentions)
Cd11b polyclonal antibodies, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Affinipure fab fragment goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Anti gfap polyclonal antibody, by Abcam (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Bovine serum albumin (bsa), by Bovogen (1 mentions)
Vectashield mounting medium containing 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
8 protocols using anti gfap monoclonal antibody
1
Immunofluorescent Labeling of GFAP in Mouse Brain
2
Multiplex Immunostaining of Neuronal Markers
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The primary antibodies that were used in this study are: anti-Gfap monoclonal antibody (Sigma), anti-Gfap polyclonal antibody (Abcam), anti-Slc1a3 (EAAT1) polyclonal antibody (Abcam), anti-Aldh1l1 polyclonal antibody (Abcam), chicken anti-Map2 antibody (Abcam), anti-Rbfox3 (NeuN) monoclonal antibody (EMD Millipore), and anti-beta III Tubulin (Tuj1) monoclonal antibody (Abcam). For mouse antibody (monoclonal antibody) on mouse tissues, the endogenous mouse IgG blocking method was used to reduce background straining. Briefly, after normal serum blocking and before primary antibody incubation, the mouse brain sections were incubated with unconjugated affiniPure Fab fragment goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch Labs, West Grove, PA) for 1 hr at room temperature. Fluorophore conjugated secondary antibodies were purchased from Invitrogen (Molecular Probe). Images were taken with a Zeiss LSM510 confocal microscope, processed with software NIH ImageJ and Imaris (Bitplane), and composed with adobe Photoshop.
3
Fluorescent Labeling of Astrocytes in Brain Slices
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Acute brain slices were fixed in 40% ethanol at 4°C for 5 min, treated with 0.1% Triton X-100 in PBS for 10 min and rinsed twice with distilled water. Preparations were incubated with 0.001% F-Jade in distilled water and gently shaken for 30 min in the dark. Later, F-Jade was removed and slices were incubated with anti-GFAP monoclonal antibody (Sigma, 1:400) diluted in 0.1% PBS-Triton X-100 with 2% NGS at 4°C overnight. After five rinses in 0.1% PBS-Triton X-100, slices were incubated with goat anti-mouse IgG Alexa Fluor 488 (1:1000) at room temperature for 50 min. After several washes, coverslips were mounted in DAKO fluorescent mounting medium and examined with a confocal laser-scanning microscope (Olympus, Fluoview FV1000, Tokyo, Japan).
4
Neuropathological Analysis of Prion Disease
5
Immunofluorescence Staining of Cultured Cells
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The cells were grown on coverslips fixed in 4% (v/v) PFA (Sigma-Aldrich) for 15 min at room temperature, or overnight at 4 °C. After rinsing in PBS, the fixed cells were permeabilized and nonspecific epitopes were blocked using 2% bovine serum albumin (Bovogen Biologicals, East Keilor, VIC, Australia) in 0.1% Tween-20/PBS, followed by incubation in the diluted primary antibody for 1 h at room temperature, or overnight at 4 °C, with an anti-GFAP monoclonal antibody (1:200, Sigma-Aldrich, cat. no. G3893) or an anti-NGFR polyclonal antibody (1:200, Santa Cruz Biotechnology, cat. no. sc8317). Following three washes in PBS, the samples were incubated for 1 h at room temperature with secondary antibodies diluted in PBS. The prepared samples were then mounted using Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) and images were captured under a fluorescence microscope (Nikon Corp., Tokyo, Japan).
6
Immunohistochemical Analysis of Glial Cells
7
Immunohistochemical Analysis of Glial Cells
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Animals were deeply anesthetized with a mixture of Xylazine and Ketamine (1:8), washed with 1X saline for 1 min and then perfused with 4% paraformaldehyde (PFA) for 15–20 min. Brains were quickly dissected out and immediately stored in 4% PFA for 24 h at 4°C, and then transferred to 30% sucrose at 4°C for 48 h. Brains were cut using a cryostat at 4°C into 20-micron-thick coronal sections and stored at -20°C. Immunohistochemistry was performed on 20 μm-thick sections. Astrocytes were probed (1:200) with monoclonal anti-GFAP antibody (Millipore Corporation, USA), and microglia were probed (1:200) with IBA-1 polyclonal antibody (Wako, USA). Dendritic cells and/or microglia were probed (1:200) with CD11b polyclonal antibodies (Thermo Fisher, USA). Nuclear staining with 4′,6-Diamidino-2-Phenylindole (DAPI) was performed according to manufacturer’s protocols (Life Technologies, USA).
8
Astrocyte Phenotyping and Scaffold Preparation
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The cultured astrocytes
on nanofibers were fixed with 4% paraformaldehyde and permeabilized
with 0.2% Triton X-100. Cells were exposed to a blocking solution
(10% horse serum and 1% bovine serum albumin) for 20 min. Then the
cells were incubated with monoclonal anti-GFAP antibody (Millipore,
Billerica, MA) to determine the cell phenotype. The secondary antibody
was Alexa Fluor 488 donkey anti-rabbit IgG (Jackson ImmunoResearch,
West Grove, PA). Nuclei were stained with diamidino-2-phenylindole.
The labeled cells were viewed using a Carl Zeiss fluorescence microscope.
To prepare the SEM test, the chemical dehydration procedure was
performed for the nanofiber scaffolds seeded with astrocytes. First,
the samples were washed with PBS and placed in 2% glutaraldehyde PBS
for 2 h. The samples were then dehydrated with graded ethanol (50,
70, 90%, and pure ethanol) followed by hexamethyldisilazane treatment.
The air-dried samples were then coated with gold before they were
analyzed by SEM.
on nanofibers were fixed with 4% paraformaldehyde and permeabilized
with 0.2% Triton X-100. Cells were exposed to a blocking solution
(10% horse serum and 1% bovine serum albumin) for 20 min. Then the
cells were incubated with monoclonal anti-GFAP antibody (Millipore,
Billerica, MA) to determine the cell phenotype. The secondary antibody
was Alexa Fluor 488 donkey anti-rabbit IgG (Jackson ImmunoResearch,
West Grove, PA). Nuclei were stained with diamidino-2-phenylindole.
The labeled cells were viewed using a Carl Zeiss fluorescence microscope.
To prepare the SEM test, the chemical dehydration procedure was
performed for the nanofiber scaffolds seeded with astrocytes. First,
the samples were washed with PBS and placed in 2% glutaraldehyde PBS
for 2 h. The samples were then dehydrated with graded ethanol (50,
70, 90%, and pure ethanol) followed by hexamethyldisilazane treatment.
The air-dried samples were then coated with gold before they were
analyzed by SEM.
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