Sections were incubated with 3% bovine serum albumin in PBS for 30 min at room temperature. Sections were then incubated in horse anti-rat immunoglobulin G (IgG) (Vector, USA; diluted 1:250) or mouse anti-GFAP antibody (Chemicon, USA; diluted 1:4,000) in PBS containing 0.3% triton X-100 overnight at room temperature. Sections were further incubated in secondary antibody and ABC complex (Jackson Immunoresearch Laboratories Inc., USA; diluted 1:250) for 2 h at room temperature. Thereafter, sections were visualized using 3,3'-diaminobenzidine in 0.1 M Tris buffer. Some sections were incubated in a mixture of mouse anti-GFAP antibody (Chemicon, USA; diluted 1:4,000)/mouse anti-SMI71 antibody (BBB marker; Covance, USA; diluted 1:1,000) in PBS containing 0.3% triton X-100 overnight at room temperature. Thereafter, sections were incubated in a mixture of FITC-conjugated secondary antiserum and Cy3-conjugated secondary antisera (Amersham, USA). The sections were washed three times for 10 min with PBS, and mounted on gelatin-coated slides. For nuclei counterstaining, we used Vectashield mounting medium with DAPI (Vector, USA).
Mouse anti gfap antibody
Manufactured by Merck Group
Sourced in United States, United Kingdom
The Mouse anti-GFAP antibody is a laboratory reagent used to detect the presence of the glial fibrillary acidic protein (GFAP) in biological samples. GFAP is a type III intermediate filament protein that is found in astrocytes and other glial cells. The antibody can be used in various immunodetection techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to identify and visualize GFAP-expressing cells.
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Lab products found in correlation
Goat anti iba1 antibody, by Abcam (3 mentions)
Mouse anti neun or goat anti map2 antibodies, by Merck Group (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Aqua poly mount, by Polysciences (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Cy3 conjugated secondary antisera, by Cytiva (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Axio observer z1 fluorescent microscope, by Zeiss (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse anticalbindin d 28k antibody, by Merck Group (1 mentions)
Vector elite abc kit, by Vector Laboratories (1 mentions)
Avidin biotin peroxidase complex, by Vector Laboratories (1 mentions)
Mouse anti nestin monoclonal antibody, by Merck Group (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Dapi staining, by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Southern Biotech (1 mentions)
Sprague dawley rat, by Charles River Laboratories (1 mentions)
Rabbit anti zo 1 antibody, by Abcam (1 mentions)
Biotinylated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Neurotrace 435 455 blue, by Thermo Fisher Scientific (1 mentions)
21 protocols using mouse anti gfap antibody
1
Immunohistochemical Staining of Brain Sections
2
Quantifying Axonal Density after Spinal Cord Injury
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At day 20 post injury, the mice were anaesthetised and perfusion-fixed with 4 % paraformaldehyde in PBS. The spinal cord tissues, including injured regions, were immersed in 30% sucrose solution. Spinal cords were sagittally sectioned at a thickness of 12 μm using a CM3050S cryostat (Leica, Heidelberg, Germany). The sections were immunostained with rabbit anti-NF-H antibody (1:1000; Cat. No. AB1989, Chemicon, Temecula, CA, United States) and mouse anti-GFAP antibody (1:1000; Cat. No. G3893, Sigma-Aldrich) as primary antibodies. Alexa Fluor 594-conjugated goat anti-mouse IgG (1:400; Cat. No. A-11005, Life Technologies) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:400; Cat. No. A-11008, Life Technologies) were used as secondary antibodies. Fluorescence images were obtained using an Axio Observer Z1 fluorescent microscope (Carl Zeiss, Oberkochen, Germany). Injured regions were defined by the GFAP-positive areas. The area of the injured region was measured using Image J analysis software (NIH, Rockville, MD, United States). Only fibre-like staining of NF-H was traced with Image J and the axonal density in each region was calculated. A blind observer analysed the images.
3
Immunohistochemical Detection of Cerebellar Cells
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For the detection of calbindin-positive and glial fibrillary acidic protein (GFAP)-positive cells in the cerebellar vermis, immunohistochemistry was performed. Briefly, the sections were incubated in PBS for 10 min and washed 3 times with PBS, and then incubated in 1% hydrogen peroxide (H2O2) for 30 min. Next, the sections were incubated overnight with mouse anti calbindin D-28k antibody (Sigma Chemical Co., St. Louis, MO, USA) at a dilution of 1:1,000 for visualization of Purkinje cells or with mouse anti-GFAP antibody (Chemicon, Temecula, CA, USA) at a dilution of 1:2,000 for visualization of astrocytes. The sections were then incubated for 1 h with anti-mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA). Bound secondary antibody was then amplified with a Vector Elite ABC kit® (Vector Laboratories). The sections were subsequently incubated with avidin-biotin-peroxidase complex (1:100; Vector Laboratories) for 1 h at room temperature. Immunoreactivity was visualized by incubating the sections in a solution consisting of 0.05% 3,3-diaminobenzidine (DAB) and 0.01% H2O2 in 50 mM Tris buffer (pH 7.6) for approximately 3 min. The sections were then mounted on gelatin-coated glass slides. The slides were air-dried overnight at room temperature, and the coverslips were mounted using Permount®.
4
Neuronal Differentiation of hUCM Cells
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To confirm the differentiation of hUCM cells into neurallike cells, the slides were fixed by 4% paraformaldehyde in 0.1 M phosphate buffer. The cells were treated by 10% H 2 O 2 to prevent non-specific antibody-antigen binding sites. The cells were then treated with the following primary antibodies at 4°C, overnight: mouse anti-nestin monoclonal antibody (1:100, Chemicon), mouse anti-NF monoclonal antibody (1:100, Chemicon), and mouse anti-GFAP antibody (1:200, Chemicon), washed with 0.1 M PBS, reacted with secondary antibodies (Envision HRP labeled; Dako); at room temperature for 1 hour, washed again with 0.1 M PBS and finally developed with diaminobenzidine (DAB). The slides were then counterstained with hematoxylin. In the control slides, all steps were processed, except the primary antibodies were omitted.
5
Primary Cultured Hippocampal Neuron Protocol
6
Astrocyte Cx30 Expression in Hippocampal CA1
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Saline and KA‐injected mice were perfused with phosphate buffered saline (PBS) 4 hr after injection and their brain rapidly removed and frozen. Cryostat brain slices were then cut and fixed for 10 min at room temperature with 4% paraformaldehyde (PFA), washed three times with PBS and pre‐incubated 1 hr with PBS‐1% gelatin in the presence of 1% Triton‐X100. Brain slices were then immunostained overnight at 4 °C for GFAP (1:500, mouse anti‐GFAP antibody, Sigma‐Aldrich) and Cx30 (1:500, rabbit anti‐Cx30 antibody, ThermoFisher) and washed in PBS three times. Appropriate secondary antibodies (goat anti‐mouse IgG conjugated to Alexa 488 and goat anti‐rabbit IgG conjugated to Alexa 561, 1:200, ThermoFisher) were finally applied for 1–2 hr at room temperature, followed by DAPI staining (1:2,000, ThermoFisher). After several washes, brain slices were mounted in fluoromount (Southern Biotechnology) and examined with a spinning‐disk confocal microscope (Eclipse Ti, Nikon) equipped with CMOS camera (Photometrics). Stacks of consecutive images were taken with a 60× objective at 500 nm intervals and acquired sequentially with 3 lasers (405, 488, and 561 nm). Z‐projections were then reconstructed using ImageJ software and average fluorescence intensity for Cx30 was measured in hippocampal CA1 stratum radiatum astrocytes.
7
Isolation and Culture of Rat Hippocampal Neurons
8
Immunohistochemical Analysis of Brain Markers
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The histological sections were subjected to antigen retrieval, cooled for 2 h, treated with 0.3% (w/v) H2O2 for 15 min, blocked with PBS containing 5% (w/v) bovine serum and 0.1% (w/v) Triton X-100 for 15 min, and incubated with a goat anti-Iba-1 antibody (1:500, Abcam, United Kingdom), rabbit anti-Zo1 antibody (1:500, Abcam, United Kingdom) or mouse anti-GFAP antibody (1:500, Sigma-Aldrich, United States) overnight at 4°C. After the sections were washed, they were incubated with biotinylated secondary antibodies (1:200, Thermo Fisher Scientific, United States) for 1 h, followed by staining with the avidin–biotin–peroxidase complex (ABC, Vector Laboratories, United States). The diaminobenzidine (DAB) reaction was visualized from the immunoprecipitated product. After the aforementioned staining procedures, the sections were dehydrated with different concentrations of ethanol and cleared with 100% xylene. Cover slips were mounted over the sections, which were then assessed under a microscope (Carl Zeiss, Oberkochen, Germany).
9
Histological Verification of Electrode Placement
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Following the final day of recording, rats were deeply anesthetized with isoflurane, and electrolytic lesions were made at the ends of tetrodes. A day or more later, rats were deeply anesthetized with isoflurane, euthanized via injection of 1mL euthasol, and perfused with 4% paraformaldehyde (PFA). The head was placed in 4% PFA. After approximately one day in PFA, the brain was extracted and placed in 4% PFA for an additional day. The brain was then transferred to a sucrose solution for approximately five days for cryoprotection, then sliced in 60–80-micron coronal sections on a cryostat. Immunohistochemistry was performed to facilitate visualization of electrode tracts. Hippocampal sections were stained with 4’,6-diamidino-2-phenylindole (DAPI) and cresyl violet or fluorescent Nissl (NeuroTrace 435/455 Blue, ThermoFisher). OFC and mPFC sections were stained to visualize glial fibrillary acidic protein (GFAP) (primary antibody: mouse anti-GFAP antibody, Sigma-Aldrich; secondary antibody: donkey anti-mouse Alexa 594 antibody, ThermoFisher), and with fluorescent Nissl and DAPI. Stained sections were visualized using fluorescence or light microscopy (Nikon Ti-E Microscope) (Extended Data Fig. 1c ). We estimated the locations of electrodes by aligning sections containing tracts to the Paxinos and Watson rat brain atlas (6th edition)43 .
10
Multimodal Neuroinflammation Analysis
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Sections were boiled in citric acid buffer and were treated with 0.3% (v/v) Triton X-100 and 10% (v/v) goat serum, they were incubated overnight at 4 °C with primary antibodies (rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody, Wako, Japan; mouse anti-NLRP3 antibody, Thermo Fisher, USA; mouse purified anti-β-amyloid, 1-42 antibody, BioLegend, USA; mouse purified anti-β-amyloid, 1-40 antibody, BioLegend, USA; rabbit anti-collagen I antibody, Abcam, USA; rat anti-LAMP2 antibody, Abcam, USA; mouse anti-GFAP antibody, Sigma, USA; rabbit anti-C3 antibody, Abcam, USA; rabbit anti-NeuN antibody, Millipore, USA; mouse anti-cleaved caspase-3 antibody, Cell Signaling Technology, USA) and then incubated with secondary antibodies. Apoptosis was detected using a transferase-mediated deoxyuridine triphosphate-biotin nick end labeling Kit (TUNEL Apoptosis Detection Kit, Roche, Switzerland). Slices were embedded using Fluoroshield™ with DAPI (Sigma, USA). Images were acquired using a Nikon fluorescence microscope (Nikon, Japan) or a confocal microscope (Leica, Germany).
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