Procedure was the same as Ki67 except without antigen retrieval. However, in double labeling combinations with nuclear proteins, antigen retrieval was performed as an inevitable part of the procedure. The primary antibodies used were polyclonal rabbit anti-glial fibrillary acidic protein at a 1:200 concentration (GFAP, Dako; Z0334) or monoclonal mouse anti-glial fibrillary acidic protein at a 1:100 concentration (GFAP, DAKO; M0761). The primary antibody used was dependent on the animal origin of the other antibody in the double label. The secondary antibodies used were Alexa-Fluor 594 goat anti-rabbit IgG at a 1:200 concentration, as above, and Alexa-Fluor 405 goat anti-mouse IgG at a 1:200 concentration. The secondary antibody used was dependent on the animal origin of the primary antibody.
Glial fibrillary acidic protein (gfap)
Manufactured by Agilent Technologies
Sourced in United States, Denmark, United Kingdom, Germany, Japan, Switzerland, Belgium, France, Spain
GFAP is a laboratory measurement for Glial Fibrillary Acidic Protein, a cytoskeletal protein found in astrocytes and other glial cells in the central nervous system. It serves as a biomarker for neural injury and disease.
Automatically generated - may contain errors
Lab products found in correlation
Neuronal nuclei (neun), by Merck Group (54 mentions)
Olig2, by Merck Group (19 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (19 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (19 mentions)
Nestin, by Merck Group (16 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (15 mentions)
Lsm 700, by Zeiss (13 mentions)
β actin, by Merck Group (10 mentions)
Lsm 710, by Zeiss (10 mentions)
Cleaved caspase 3, by Cell Signaling Technology (9 mentions)
βiii tubulin, by Merck Group (9 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (9 mentions)
Olig2, by R&D Systems (8 mentions)
Nestin, by Abcam (8 mentions)
Alexa 488, by Thermo Fisher Scientific (8 mentions)
Abc kit, by Vector Laboratories (7 mentions)
Iba 1, by Abcam (7 mentions)
Ubiquitin, by Agilent Technologies (7 mentions)
Gapdh, by Merck Group (7 mentions)
Mab377, by Merck Group (7 mentions)
509 protocols using glial fibrillary acidic protein (gfap)
1
Double Labeling of GFAP in Cells
2
Immunostaining of NHP Cerebellar Tissue
3
Immunofluorescence Profiling of PDGF Signaling
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Double immunofluorescence was performed using the following antibody combinations: PDGF-C and GFAP (1:25; Dako, Denmark), CD68 (1:25; Epitomics, USA), hGLUT5 (1:50; IBL, Japan), or CD45 (1:50; Epitomics); PDGF-D and GFAP, CD68, hFLUT5, or CD45; PDGFR-α and GFAP, CD68, hGLUT5, or CD31 (1:20; Dako, Denmark); and PDGFR-β and GFAP, CD68, hGLUT5, or CD31.
GFAP, CD68, hGLUT5, CD45, and CD31 were adopted as markers for astrocytes, monocytes, microglia, lymphocytes, and endothelial cells, respectively. All sections were incubated with their respective antibodies for 24 hours with CD68, hGLUT5, and GFAP, and for 48 hours with PDGF-A, B, C, D, and PDGFR-α and β. Then, after washing the primary antibodies, Alexa Fluor 488 (1:25; Molecular Probes, USA) or Alexa Fluor 546 (1:25; Molecular Probes) was used (Table3 ). Finally, the sections were examined using an LSM510 laser scanning confocal microscope (Carl Zeiss, Germany).
GFAP, CD68, hGLUT5, CD45, and CD31 were adopted as markers for astrocytes, monocytes, microglia, lymphocytes, and endothelial cells, respectively. All sections were incubated with their respective antibodies for 24 hours with CD68, hGLUT5, and GFAP, and for 48 hours with PDGF-A, B, C, D, and PDGFR-α and β. Then, after washing the primary antibodies, Alexa Fluor 488 (1:25; Molecular Probes, USA) or Alexa Fluor 546 (1:25; Molecular Probes) was used (Table
4
Protein Expression Analysis in Neurodegeneration
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The following antibodies were used for Western blot: Progranulin (R&D Systems #AF2420, 1:500 and MilliporeSigma #HPA008763, 1:500), NeuN (MilliporeSigma #MAB377, 1:500), GFAP (Agilent #Z033429, 1:500), LC3B (MilliporeSigma #L7543, 1:500), p62 (Proteintech #18420-1-AP, 1:1000), LAMP-1 (Santa Cruz Biotechnology #sc-20011, 1:1000), GM130 (Cell Signaling Technologies #12480, 1:1000), Grp94 (Santa Cruz Biotechnology #sc-32249, 1:500), Cytochrome C (Santa Cruz Biotechnology #sc-13156, 1:500), and Gapdh (MilliporeSigma #MAB374, 1:5000). Antibodies used for immunostaining included: Progranulin (R&D Systems #AF2420, 1:500), LAMP-2 (Invitrogen # PA1655, 1:500), GFP (Cell Signaling Technologies #2956, 1:500), Cathepsin D (R&D Systems #AF1029, 1:500), NeuN (MilliporeSigma #MAB377, 1:500), GFAP (Agilent #Z033429, 1:1000), and MAP2 (Invitrogen # PA110005).
5
Quantifying Inflammatory Mediators and Cellular Signaling
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Standard dual-antibody solid-phase immunoassays were performed according to the manufacturer's instructions (ELISA Development Kit, Peprotech) to quantify chemokine/cytokine [interferon gammainduced protein 10, IP-10/CXCL10; monocyte chemoattractant protein-1, MCP-1/CCL2; interleukin-6, IL-6] levels in plasma and tissues. Absorbance of the nal product was read on a BIOTEK Synergy 2 spectrophotometer at 450 nm using a Gen5 microplate reader and imager software by BIOTEK.
Western blot analysis was used to measure NFκB-p65, p38 MAPK, and glial brillary acidic protein (GFAP) levels in tissue homogenates. Beta-tubulin was also measured for normalization. Total protein (100 µg) was analyzed by SDS polyacrylamide gel electrophoresis (PAGE) and transferred to the PVDF membrane. The membrane was incubated in target-speci c 1° antibody [p65 (1:1000), Cat# 4764S; p38 (1:1000), Cat# 9212S; GFAP (1:1000), Cat#3670S; β-tubulin (1:1000), Cat# 2146S; Cell Signaling Technology], washed 6 times in Tris-buffered saline with 0.1% Tween (TBST), then incubated in 2°a ntibody [Goat-anti-Rabbit IgG (1:10,000), Cat# 925-32211; Donkey-anti-Mouse IgG (1:10,000), CAT# 926-32212; Li-Cor). Licor-CLX Odyssey was used to image Western blots, and protein signals were analyzed using Image J for relative quanti cation.
Western blot analysis was used to measure NFκB-p65, p38 MAPK, and glial brillary acidic protein (GFAP) levels in tissue homogenates. Beta-tubulin was also measured for normalization. Total protein (100 µg) was analyzed by SDS polyacrylamide gel electrophoresis (PAGE) and transferred to the PVDF membrane. The membrane was incubated in target-speci c 1° antibody [p65 (1:1000), Cat# 4764S; p38 (1:1000), Cat# 9212S; GFAP (1:1000), Cat#3670S; β-tubulin (1:1000), Cat# 2146S; Cell Signaling Technology], washed 6 times in Tris-buffered saline with 0.1% Tween (TBST), then incubated in 2°a ntibody [Goat-anti-Rabbit IgG (1:10,000), Cat# 925-32211; Donkey-anti-Mouse IgG (1:10,000), CAT# 926-32212; Li-Cor). Licor-CLX Odyssey was used to image Western blots, and protein signals were analyzed using Image J for relative quanti cation.
6
Quantifying Inflammatory Mediators and Cellular Signaling
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Standard dual-antibody solid-phase immunoassays were performed according to the manufacturer's instructions (ELISA Development Kit, Peprotech) to quantify chemokine/cytokine [interferon gammainduced protein 10, IP-10/CXCL10; monocyte chemoattractant protein-1, MCP-1/CCL2; interleukin-6, IL-6] levels in plasma and tissues. Absorbance of the nal product was read on a BIOTEK Synergy 2 spectrophotometer at 450 nm using a Gen5 microplate reader and imager software by BIOTEK.
Western blot analysis was used to measure NFκB-p65, p38 MAPK, and glial brillary acidic protein (GFAP) levels in tissue homogenates. Beta-tubulin was also measured for normalization. Total protein (100 µg) was analyzed by SDS polyacrylamide gel electrophoresis (PAGE) and transferred to the PVDF membrane. The membrane was incubated in target-speci c 1° antibody [p65 (1:1000), Cat# 4764S; p38 (1:1000), Cat# 9212S; GFAP (1:1000), Cat#3670S; β-tubulin (1:1000), Cat# 2146S; Cell Signaling Technology], washed 6 times in Tris-buffered saline with 0.1% Tween (TBST), then incubated in 2°a ntibody [Goat-anti-Rabbit IgG (1:10,000), Cat# 925-32211; Donkey-anti-Mouse IgG (1:10,000), CAT# 926-32212; Li-Cor). Licor-CLX Odyssey was used to image Western blots, and protein signals were analyzed using Image J for relative quanti cation.
Western blot analysis was used to measure NFκB-p65, p38 MAPK, and glial brillary acidic protein (GFAP) levels in tissue homogenates. Beta-tubulin was also measured for normalization. Total protein (100 µg) was analyzed by SDS polyacrylamide gel electrophoresis (PAGE) and transferred to the PVDF membrane. The membrane was incubated in target-speci c 1° antibody [p65 (1:1000), Cat# 4764S; p38 (1:1000), Cat# 9212S; GFAP (1:1000), Cat#3670S; β-tubulin (1:1000), Cat# 2146S; Cell Signaling Technology], washed 6 times in Tris-buffered saline with 0.1% Tween (TBST), then incubated in 2°a ntibody [Goat-anti-Rabbit IgG (1:10,000), Cat# 925-32211; Donkey-anti-Mouse IgG (1:10,000), CAT# 926-32212; Li-Cor). Licor-CLX Odyssey was used to image Western blots, and protein signals were analyzed using Image J for relative quanti cation.
7
Multimodal Tissue Analysis of Organ Fixation
8
Multimodal Tissue Analysis of Organ Fixation
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Harvested organs were fixed in 4% paraformaldehyde (PFA) for 24 hours or IHC zinc fixative (BD Pharmingen) for anti-perforin stained tissues. Fixed tissues were then sucrose sedimented in 10%, 20% and 30% steps prior to embedding within optimum cutting temperature (O.C.T.) medium (Tissue Tek). Frozen sections of 5–7 μm thicknesses were cut and left to dry at ambient temperature. These tissues were then stained with primary antibodies: Cleaved Caspase 3 (Cell Signaling Technology), CD31 (Novus Biologicals INC), CD45 (Novus Biologicals INC), glial fibrillary acidic protein (GFAP) (Agilent Technologies INC), rabbit-neuronal marker (NeuN) (Cell Signaling Technology), guinea pig-NeuN (EMD Millipore Corporation), rat anti-perforin (Novus Biologicals INC) and anti-ZIKV rat serum 22 (link) after rehydration with PBS (2X for 10 minutes) and permeabilization with PBST (3X for 10 minutes) at RT overnight. Slides were washed and incubated with appropriate secondary prior to a final wash series and incubation with 4′,6-diamidino-2-phenylindole (DAPI) prior to mounting with SlowFade Diamond Antifade (Fisher Scientific Company LLC). Stained tissues were then analyzed by fluorescence microscopy (BX51; Olympus) or confocal microscopy (TCS SP2; Leica).
9
Comprehensive Lipid and Protein Analysis
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Methanol, acetonitrile, trifluoroacetic acid (TFA), sodium trifluoroacetate (NaTFA), 50 mM triethylammonium bicarbonate (TEAB), bicinchoninic acid (BCA) protein assay kit, Optimal Cutting Temperature (OCT) embedding medium, 10% neutral-buffered formaldehyde, and hematoxylin and eosin (H&E) were purchased from Thermo Fisher Scientific (Waltham, MA). The internal standards for LC-ESI-MS including galactosyl(ß) ceramide (d18:1/12:0) and mono-sulfo galactosyl(ß) ceramide (d18:1/17:0) were purchased from Avanti Polar Lipids (Alabaster, AL). Bis-cyclohexanone oxaldihydrazone (Cz), ammonium bicarbonate, dichloromethane, 1, 5-diaminonapthalene (DAN), Urea, and luxol fast blue (LFB) were obtained from Sigma-Aldrich (St. Louis, MO). The following rabbit polyclonal primary antibodies were used: glial fibrillary acidic protein (GFAP; Agilent Technologies, Carpinteria, CA), ionized calcium-binding adapter molecule 1 (IBA1; Wako Chemicals, Richmond, VA), and PLP (Sigma-Aldrich, St. Louis, MO). Antirabbit horseradish peroxidase (HRP) secondary antibody was purchased from Sigma-Aldrich (St. Louis, MO).
10
Western Blotting Analysis of Cellular Proteins
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Total proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane for Western blotting. The membranes were blocked with 5% skim milk (Sigma) in Tris–buffered saline for 1 h at room temperature and then incubated with various primary antibodies overnight at 4°C: tumor necrosis factor (TNF)-α, cluster of differentiation 11b (CD11b), heme oxygenase (HO)-1, ferritin, and tubulin (all 1:1,000; Abcam, Cambridge, MA, United States); NAD(P)H quinone dehydrogenase 1 (NQO1), B-cell lymphoma-2 (Bcl-2)-associated X protein (BAX), transferrin, and actin (all 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States); p62 and microtubule-associated protein 1A/1B light chain (LC) 3B (all 1:1,000; Cell Signaling Technology, Danvers, MA, United States); and glial fibrillary acidic protein (GFAP) (1:5,000; Agilent Technologies, Santa Clara, CA, United States). Further, the blots were probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Santa Cruz Biotechnology) and visualized using SuperSignal West Femto Substrate Maximum Sensitivity Substrate (Thermo Fisher Scientific). A ChemiDoc image analyzer was used to detect immunoblotted bands (Bio-Rad, Hercules, CA, United States).
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