Mice were transcardially perfused with 20 mL of cold saline followed by 20 mL of 4% paraformaldehyde. The brain was then removed and postfixed in the same fixative for 24 hours, followed by immersion in 30% sucrose at 4°C for 2 to 3 days. Coronal sections (30 μm) from the area located around 2.06 mm posterior to bregma were subsequently obtained using a cryostat. Sections were rinsed in PBS 3 times, and then blocked with 2% goat serum in PBS containing 0.2% Triton X‐100 for 2 hours at room temperature. Next, the sections were incubated overnight at 4°C with the primary antibodies: rabbit anti‐GFAP (glial fibrillary acidic protein) (1:300; Millipore Burlington, MA) and rabbit anti‐Iba1 (ionized calcium‐binding adaptor molecule 1) (1:300; Wako, Richmond, VA). After extensive washing, the sections were incubated with fluorescent secondary antibodies for 2 hours at room temperature. Images were captured on a Zeiss fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
Rabbit anti gfap glial fibrillary acidic protein
Manufactured by Merck Group
Sourced in United States
Rabbit anti-GFAP (glial fibrillary acidic protein) is a primary antibody that specifically binds to GFAP, an intermediate filament protein found in astrocytes and other glial cells. It is commonly used as a marker for the identification and visualization of astrocytes in various research and clinical applications.
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Lab products found in correlation
Rabbit anti iba1, by Fujifilm (2 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
Cryostat, by Leica (1 mentions)
Rabbit anti nestin, by Merck Group (1 mentions)
Mouse anti microtubule associated protein 2 map 2, by Merck Group (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Alexa fluor 488 antibody, by Abcam (1 mentions)
Cm1900, by Leica (1 mentions)
Thioflavin s, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
4 protocols using rabbit anti gfap glial fibrillary acidic protein
1
Immunohistochemical Analysis of Glial Cells
2
Immunofluorescence Staining of Mouse Brain
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Immunofluorescence staining was performed on frozen tissues, as described previously (Jiang et al., 2017 (link)). In short, mouse brains were fixed by transcardial perfusion with 4% paraformaldehyde, and stored at −80°C. Frozen brain sections (35 μm thick) were obtained using a Leica cryostat, and immunostained using a free-floating staining method with the following primary antibodies: rabbit anti-GFAP (glial fibrillary acidic protein; 1:500; Millipore, Burlington, MA, USA) and rabbit anti-Iba1 (ionized calcium-binding adaptor molecule 1; 1:500; Wako, Richmond, VA, USA). Images were captured on a Zeiss Axio Imager Z2 motorized fluorescence microscope (Carl Zeiss MicroImaging). The striatal areas of the ipsilateral and contralateral hemispheres were measured and calculated as the percentage of ipsilateral/contralateral striatal area. GFAP+ astrocytes, Iba1+ microglia, and lectin-stained vessels were analyzed in a blinded fashion by measuring fluorescence intensity in four defined regions of interest (ROI) in the ipsilateral or contralateral cortex and striatum (size: 500 × 500 μm; ROI centered 1.75 mm lateral to midline/0.75 mm, 2.5 mm, 3.5 mm, and 4.0 mm below brain surface). The mean values of GFAP and Iba1 fluorescence intensity and vascular density were calculated as the percentage of ipsilateral/contralateral ROI.
3
Immunocytochemical Analysis of Neural Cell Markers
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Immunocytochemistry experiment was performed as described previously (Noureddini et al., 2012). Briefly, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100. After blocking with 3% goat serum albumin, cells were incubated with primary antibodies for glial, neuronal and pre-neuronal markers at 37°C for 12 hours. The following primary antibodies and dilutions were used: mouse anti-β-tubulin-Tuj1 (1:500; Chemicon, Billerica, MA, USA); rabbit anti-glial fibrillary acidic protein (GFAP; 1:500; Sigma); rabbit anti-nestin (1:1,000; Sigma); mouse anti-microtubule-associated protein 2 (MAP-2; 1:500; Sigma). Then the cells were washed with PBS and reacted with the fluorescent isothiocyanate (FITC) conjugated secondary antibodies against rabbit and mouse Fc region (Sigma; 1:500) at room temperature for 2 hours. Finally, the cells were washed with PBS three times, and 4′,6-diamidino-2-phenylindole (DAPI) was used for DNA staining. The cells were visualized with Ceti immunofluorescence microscopy (Belgium).
4
Immunohistochemical Analysis of Amyloid Plaques
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Animals were deeply anesthetized, killed, and perfused intracardially with normal saline and fixed with 4% paraformaldehyde (PFA). Brains were placed in 15% sucrose in PBS for 6-12 h, then 30% sucrose in PBS until equilibrated. Brain coronal sections (10 μm) were cut with a cryostat (Leica CM1900), sections were used for immunohistochemistry. Slices were washed with PBS and fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100, then pre-incubated in 1% BSA solution for blocking. Slices were incubated overnight at 4 °C with the primary antibody solution (rabbit anti-amyloid-β antibody, Abcam; 1:500), mouse anti-NeuN (1:100, Sigma), rabbit anti-glial fibrillary acidic protein (GFAP) (1:250, Sigma) on a shaker. After washing with PBS, the sections were incubated with the secondary antibody (goat anti-rabbit Alexa Fluor 488 antibody, Abcam; 1:1000; goat anti-mouse Alexa Fluor 488 antibody, Abcam; 1:1000) for 2 h at room temperature. Sections were mounted in the vectashield mounting medium with DAPI (Vector laboratories). For the detection of amyloid plaques, brain tissue sections were stained in 0.1% thioflavin-S (Sigma) and rinsed with 70% ethanol. The brain tissue sections were also stained with hematoxylin and eosin (H&E) to assess the histological damage.
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