Rabbit anti gfap

Manufactured by Thermo Fisher Scientific

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Rabbit anti-GFAP is a primary antibody that specifically binds to the Glial Fibrillary Acidic Protein (GFAP), an intermediate filament protein found in astrocytes and other glial cells. This antibody can be used to identify and analyze glial cells in various research and diagnostic applications.

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Anti-GFAP (49 citations) Rabbit anti-mouse GFAP (2 citations)

14 protocols using rabbit anti gfap

Immunohistochemical Analysis of Astrocytes and Microglia

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Immunohistological studies were performed using 30 µm-thick frozen brain coronal sections that were obtained through cryo-sectioning and preserved in phosphate-buffered saline (PBS) 1× until analysis. After the treatment with citrate buffer (0.01 M citric acid, 0.05% Tween 20, pH 6.0) at 70 °C for 50 min, samples were washed thoroughly with a solution of 2% immunoglobulin (Ig)G-free albumin (Sigma, St Louis, MO, USA) in TBS for 20 min at room temperature. Brain sections were then incubated overnight at 48 °C with either rabbit anti-GFAP (Invitrogen, Carlsbad, CA, USA) or anti-Iba-1 (Wako Chemicals Inc., Richmond, VA, USA) in TBS-2% BSA to detect astrocytes and microglia, respectively. After washing, sections were incubated for 1 h at room temperature with AlexaFluor 594 goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) diluted in TBS-2% BSA. Sections were mounted onto glass slides in Vectashield medium (Vector Laboratories, Burlingame, CA, USA) containing 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei The mean fluorescence intensity was quantified using Image J software (National Institute of health, Bethesda, MD, USA). Three mice for each experimental condition were analyzed.

Giraldo-Velásquez M.F., Pérez-Osorio I.N., Espinosa-Cerón A., Bárcena B.M., Calderón-Gallegos A., Fragoso G., Torres-Ramos M., Páez-Martínez N, & Sciutto E. (2022). Intranasal Methylprednisolone Ameliorates Neuroinflammation Induced by Chronic Toluene Exposure. Pharmaceutics, 14(6), 1195.

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Immunofluorescence Staining of Mouse Brain Sections

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The half of each brain obtained as described above was fixed in 4 % buffered formalin at 4 °C overnight. Free-floating 30 μm-thick mouse brain sections were processed as previously described [33 (link)]. After antigen retrieval by incubating in citrate buffer (0.01 M citric acid, 0.05 % Tween 20, pH 6.0) at 70 °C for 50 min, samples were thoroughly washed several times with TBS and blocked with a solution of 2 % IgG-free albumin (Sigma) in TBS for 20 min at room temperature. Brain sections were then incubated overnight at 4 °C with rabbit anti-GFAP (Invitrogen) or anti-Iba-1 (WAKO, VA) polyclonal antibody in TBS-2 % BSA to detect astrocytes or microglia, respectively. After washing, sections were incubated for 1 h at room temperature with AlexaFluor 594 goat anti-rabbit IgG (Molecular Probes, OR) diluted in TBS-2 % BSA. Samples were mounted onto glass slides in Vectashield medium (Vector Laboratories, CA) containing DAPI for nuclei imaging. Samples were viewed on an Olympus Ix51 microscope equipped with a DP71 camera (Nikon Instruments Inc., NY).

Meneses G., Bautista M., Florentino A., Díaz G., Acero G., Besedovsky H., Meneses D., Fleury A., Del Rey A., Gevorkian G., Fragoso G, & Sciutto E. (2016). Electric stimulation of the vagus nerve reduced mouse neuroinflammation induced by lipopolysaccharide. Journal of Inflammation (London, England), 13, 33.

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Immunohistochemical Staining of Mouse Brain

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Mice were anesthetized and perfused with PFA 4%. Brains were dissected, embedded in optimal cutting temperature (OCT, Tissue-Tek) and sectioned (10 μm) using a cryostat. For immunohistochemistry, slides underwent citrate buffer antigen retrieval and were then incubated with blocking solution (5% goat serum, 1% BSA, 0.05% Triton-X in PBS) for 1 hr. Then, slides were incubated overnight at 4°C with the following primary antibodies: rabbit anti -GFAP (1:200, Invitrogen), rabbit anti-DARPP-32 (1:50, Abcam), and mouse anti-TH (1:200, Sigma). Then, sections were incubated with the secondary antibody Alexa Fluor 488 and 568 (1:500; Invitrogen) for 1 hr. The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; 1:1000; Sigma-Aldrich).

Glat M.J., Ben-Zur T., Barhum Y, & Offen D. (2016). Neuroprotective Effect of a DJ-1 Based Peptide in a Toxin Induced Mouse Model of Multiple System Atrophy. PLoS ONE, 11(2), e0148170.

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Immunofluorescence Staining of Cells and Brain Tissue

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Cell culture on coverslip were fixed with 4% PFA for 8min
and quenched with 10mM Glycine for 30min and then blocked in blocking buffer
(0.02% Triton X-100 and 5% normal donkey serum in PBS) for
20min and incubate with primary antibodies for 40min at room temperature.
Cryosections (12-μm thick) or vibratome sections (50-μm
thick) were permeabilized and blocked in blocking buffer (0.3%
Triton X-100 and 5% normal donkey serum in PBS) for 1 h at room
temperature and overlaid with primary antibodies overnight at 4 °C.
Antibodies used in the study were: rabbit anti-Olig2 (Millipore, AB9610),
rat anti-PDGFRα (BD Bioscience, 558774), mouse anti-APC (CC1,
Oncogene Research, OP80), goat anti-MBP (Santa Cruz Biotechnology,
sc-13914), rabbit anti-CHD8 (Abcam, ab84527), rabbit anti-GFAP (Invitrogen,
#13-0300), rabbit anti-cleaved caspase 3 (Cell Signaling Technology,
#9661), rabbit anti-GFP (Molecular Probes, #A-11122), Rabbit
Monoclonal anti-Sox10 (Abcam, ab180862), mouse anti-NeuN (Millipore,
MAB377). After washing with 0.2% Triton X-100 in PBS, cells or brain
sections were incubated with secondary antibodies conjugated to Cy2, Cy3 or
Cy5 (Jackson ImmunoResearch Laboratories) for 2 h at room temperature,
stained in DAPI for 5 min, washed in PBS and mounted with Fluoromount-G
(SouthernBiotech). Cell counting was carried out in a double-blind
manner.

Zhao C., Dong C., Frah M., Deng Y., Marie C., Zhang F., Xu L., Ma Z., Dong X., Lin Y., Koenig S., Nait-Oumesmar B., Martin D.M., Wu L.N., Xin M., Zhou W., Parras C, & Lu Q.R. (2018). Dual-requirement of CHD8 for Chromatin Landscape Establishment and Histone Methyltransferase Recruitment to Promote CNS Myelination and Myelin Repair. Developmental cell, 45(6), 753-768.e8.

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Spinal Cord Immunofluorescence Staining

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Spinal cord sections were processed as above. For immunofluorescence, slides were first desiccated for at least two hours and blocked with 5% Normal Donkey Serum with or without 0.3% Triton-X 100. Primary antibodies were incubated overnight at 4°C: goat anti-CXCL1 1:50 (R&D Systems, Minneapolis, MN), rabbit anti – GFAP 1:500 (Life Technologies, Carlsbad, CA), rat anti – Ly-6B.2 1:100 (Serotec, Raleigh, NC), rabbit anti - Iba11:500 (Wako Chemicals, Richmond, VA), rabbit anti – GST-π 1:1000 (MBL, Woburn, MA).

Marro B.S., Grist J.J, & Lane T.E. (2016). Inducible Expression of CXCL1 within the Central Nervous System Amplifies Viral-Induced Demyelination. The Journal of Immunology Author Choice, 196(4), 1855-1864.

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Fluorescence Immunostaining of Retinal Cells

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Retinas were prepared as described for IB₄-Alexa 594 staining, and incubated with primary antibodies in RSB at 4°C overnight. Primary antibodies included rabbit anti-GFAP (1∶200, Life Technologies), rat anti-GFAP (2 µg/ml, Life Technologies), rabbit anti-Pax2 (1 µg/ml, Life Technologies), and goat anti- PDGFRα (1 µg/ml, R&D Systems). For Pax2 and GFAP double IF staining, rabbit anti-Pax2 and rat anti-GFAP were used. Following incubation with primary antibodies, retinas were washed, and incubated overnight with appropriate secondary antibodies including goat anti-rabbit IgG-Alexa fluor®-488 (1∶200, Life Technologies), donkey anti-rat IgG-Cy3 (2 µg/ml, Jackson ImmunoResearch, , West Grove, PA), and donkey anti-goat IgG-Alexa fluor®-488. Stained retinas were washed thoroughly, and mounted in 50% glycerol in PBS. Images were taken with a Zeiss LSM 510 confocal microscope.

Duan L.J., Takeda K, & Fong G.H. (2014). Hypoxia Inducible Factor-2α Regulates the Development of Retinal Astrocytic Network by Maintaining Adequate Supply of Astrocyte Progenitors. PLoS ONE, 9(1), e84736.

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Quantification of Inflammatory Cells in Spinal Cord

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Spinal cord sections were processed as described above. For immunofluorescence, slides were first desiccated for two hours and blocked with 5% Normal Donkey Serum with or without 0.3% Triton‐X 100. Primary antibodies were incubated overnight at 4°C: goat anti‐CXCL1 1:50 (R&D Systems, Minneapolis, MN), rabbit anti‐GFAP 1:500 (Life Technologies, Carlsbad, CA), and rat anti‐Ly6B.2 1:100 (Serotec, Raleigh, NC). Images were analyzed using the Image J software (NIH) according to previously described methods 61, 62. Quantification of Ly6B.2‐positive cells within spinal cords of experimental mice was determined by counting cells in a minimum of five spinal cord sections/per mouse with a minimum of three mice per group.

Grist J.J., Marro B.S., Skinner D.D., Syage A.R., Worne C., Doty D.J., Fujinami R.S, & Lane T.E. (2018). Induced CNS expression of CXCL1 augments neurologic disease in a murine model of multiple sclerosis via enhanced neutrophil recruitment. European Journal of Immunology, 48(7), 1199-1210.

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Spinal Cord Immunofluorescence Analysis

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Spinal cord sections were processed as described above. For
immunofluorescence, slides were first desiccated for two hours and blocked with
5% Normal Donkey Serum with or without 0.3% Triton-X 100.
Primary antibodies were incubated overnight at 4°C: goat anti-CXCL1 1:50
(R&D Systems, Minneapolis, MN), rabbit anti-GFAP 1:500 (Life
Technologies, Carlsbad, CA), and rat anti-Ly6B.2 1:100 (Serotec, Raleigh, NC).
Images were analyzed using the Image J software (NIH) according to previously
described methods [62 (link), 63 (link)]. Quantification of
Ly6B.2-positive cells within spinal cords of experimental mice was determined by
counting cells in a minimum of 5 spinal cord sections/per mouse with a minimum
of 3 mice per group.

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Multimodal Analysis of Neuroinflammation

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Immunofluorescence microscopy was performed on 35-μm brain slices containing the region of interest (see Additional file 1: Figure S1a). The following primary antibodies were used: rabbit-anti Iba-1 (1:500, Wako); rabbit anti-GFAP (1:250, Thermo Fisher); mouse monoclonal anti-NeuN (1:250, Millipore); goat anti-IL-13 (1:50, R&D system), rabbit anti-3-Nitrotyrosine (3-NT, 1:200, Millipore). After washing, a combination of secondary antibodies was used for detection: 488 Alexa goat anti-rabbit, 594 Alexa goat anti-mouse IgG1 (Invitrogen). Slides were counterstained with DAPI and were cover-slipped in Fluoromount. Digital images were taken using a Zeiss LSM 710 laser scanning confocal microscope (LSCM). Tyrosine Hydroxylase DAB Immunohistochemical analysis was performed using rabbit anti-Tyrosine Hydroxylase, (1:10000; Millipore), incubated with a biotinylated goat-anti rabbit antibody (1:400; Vector Labs) for 1 h at RT and then stained with Vectastain ABC Kit (Standard) and DAB Peroxidase Substrate (Vector Labs). For cresyl violet (CV) staining (Nissl), adjacent sections were mounted on positive charged slides and air-dried. The slides were soaked in CV solution (0.25% cresyl violet and 0.3% acetic acid) for 15 min (warmed at 50 °C), were dehydrated with alcohol and xylene, and were cover-slipped with Vectamount.

Mori S., Sugama S., Nguyen W., Michel T., Sanna M.G., Sanchez-Alavez M., Cintron-Colon R., Moroncini G., Kakinuma Y., Maher P, & Conti B. (2017). Lack of interleukin-13 receptor α1 delays the loss of dopaminergic neurons during chronic stress. Journal of Neuroinflammation, 14, 88.

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Immunoblotting and Immunofluorescence Analysis

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The following primary antibodies were used for immunoblotting (IB) and immunofluorescence (IF). Rat anti‐Serpin A1a (#MAB7690, IB, IF), rat anti‐Serpin C1 (#MAB1287, IB, IF, both from R&D Systems, Minneapolis, MN), mouse anti‐UBQLN2 #NBP2‐25164, IB, IF, Novus Biologicals, Littleton, CO), mouse anti‐neuroserpin (#66997‐1, IB, IF, Proteintech Group, Inc, Rosemont, IL), mouse anti‐ubiquitin (sc‐8017, Santa Cruz Biotechnology, IB, Santa Cruz, CA), Rabbit anti‐GFAP (#PA1‐10019 IF, Thermo Fisher Scientific), rabbit anti‐IBA1 (#019‐19741, IF, FUJIFILM Wako Pure Chemicals, Corp, Richmond, VA), rabbit anti‐Serpin B1 (#PA576875, IB, IF, Thermo Fisher Scientific), rabbit anti‐Serpin C1/antithrombin III (#NBP2‐76966, IB, Novus Biologicals), rabbit anti‐LC3A/B (#4108, IF, Cell Signaling Technology, Danvers, MA, USA), rabbit anti‐Myc (UMY81, IB, home‐made against a peptide with an amino acid sequence of MEQKLISEEDLN), rabbit anti‐Neuroserpin (#ab33077, IB, IF, abcam, Eugene, OR), rabbit anti‐p97/VCP (UMY475, IB, home‐made), rabbit anti‐UBQLN2 (#23449‐1‐AP, IF Proteintech Group, Inc.), goat anti‐ChAT (#AB144P, IF, Millipore‐Sigma, Burlington, MA), and goat anti‐LAMP1 (sc‐8098, IF, Santa Cruz Biotechnology (Dallas, TX, USA).

Higgins N.R., Greenslade J.E., Wu J.J., Miranda E., Galliciotti G, & Monteiro M.J. (2021). Serpin neuropathology in the P497S UBQLN2 mouse model of ALS/FTD. Brain Pathology, 31(5), e12948.

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