Brain sections were processed for immunofluorescence staining as previously described [46 (link)] and reported below. In brief, all sections were incubated overnight (O/N) in a humidified chamber at 37 °C using the following primary antibodies: anti-GFAP antibody (1:100; sc-33673; Santa Cruz Biotechnology), anti-IBA1 antibody (1:100; sc-32725; Santa Cruz Biotechnology). Sections were washed with PBS solution and incubated with IgG (H + L) highly cross-adsorbed goat anti-mouse secondary antibody, Alexa Fluor™ (1:1000 in PBS v/v, Molecular Probes, Altrincham, UK) for 1 h at 37 °C. After washing in PBS, nuclear staining with 4′,6′-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) (2 µg/mL) in PBS was added. Slides were observed and photographed at 40× magnifications using a Leica DM2000 microscope (Leica QWin V3, Cambridge, UK).
Anti gfap antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-GFAP antibody is a laboratory reagent used for the detection and analysis of the Glial Fibrillary Acidic Protein (GFAP) in biological samples. GFAP is an intermediate filament protein that is primarily expressed in astrocytes and other glial cells in the central nervous system. The Anti-GFAP antibody can be utilized in various experimental techniques, such as immunohistochemistry, western blotting, and flow cytometry, to study the expression and localization of GFAP in different cell types and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti iba1 antibody, by Santa Cruz Biotechnology (2 mentions)
Sc 33673, by Santa Cruz Biotechnology (1 mentions)
Sc 32725, by Santa Cruz Biotechnology (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Sybr green pcr master mix, by Promega (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Merck Group (1 mentions)
Annexin 5, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Vectastain universal elite abc kit, by Vector Laboratories (1 mentions)
Anti cox 2 antibody, by Santa Cruz Biotechnology (1 mentions)
Dm6 microscope, by Leica (1 mentions)
Las x navigator software, by Leica (1 mentions)
Anti mpo antibody, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated bovine anti mouse igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
Biotin conjugated goat anti mouse igg and avidin biotin peroxidase complex, by Vector Laboratories (1 mentions)
Eclipse e200 led microscope, by Nikon (1 mentions)
Vitronectin, by Merck Group (1 mentions)
7 protocols using anti gfap antibody
1
Immunofluorescence Staining of Brain Sections
2
Primary Astrocyte Isolation and Culture
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Primary astrocytes were prepared according to previous study with modifications [23 (link), 24 (link)]. Briefly, cortex was dissected from postnatal day 0-1 C57BL/6 mouse pups. Cells were seeding in normal glucose Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS). Cells were then cultured CO2 incubators with medium changed every 2-3 days. When cells became confluent, plate was shaken on an orbital shaker. Then, medium was removed, and cells were split into new plates and culture for 2-7 days. These cells were then seeded and used for experiments. Propidium iodide (PI), annexin V, rotenone, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and oligomycin were purchased from Sigma. Anti-GFAP antibody and anti-actin antibody were purchased from Santa Cruz; all the other antibodies were purchased from Cell Signaling. Quantitative real-time PCR was carried out using SYBR Green PCR Master Mix (Promega) and 7300 Real-Time PCR System from Applied Biosystems.
3
GFAP Immunohistochemistry in Rabbit
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Rabbit polyclonal anti-GFAP antibody (1:1000; Santa Cruz biotechnology, Inc., USA) was used, with a 3-h incubation at 37 °C, on a Vectastain Universal Elite ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions (counterstained with haematoxylin). Endogenous peroxidase was inactivated by incubation with 3% hydrogen peroxide for 5 min. For the control study to confirm the specificity of immunostaining, phosphate buffered saline (PBS) or normal rabbit serum was substituted for the primary antibody.
4
Immunohistochemical Analysis of Brain Markers
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Immunohistochemical analysis was performed as already described [50 (link)]. Subsequently, the sections were incubated overnight with an anti-Bromodeoxyuridine (BrdU) antibody (1:100; Santa Cruz Biotechnology) or anti-MPO antibody (1:250; Santa Cruz Biotechnology) or anti-NRLP3 antibody (1:250; Santa Cruz Biotechnology) or anti-COX-2 antibody (1:250; Santa Cruz Biotechnology) or anti-Iba-1 antibody (1:250; Santa Cruz Biotechnology) or anti-GFAP antibody (1:450; Santa Cruz Biotechnology). Sections were washed with PBS and incubated with peroxidase-conjugated bovine anti-mouse IgG, secondary antibody (1:2000 Jackson Immuno Research, West Grove, PA, USA). Specific labeling was provided with a biotin-conjugated goat anti-mouse IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Images were collected using a Leica DM6 microscope associated with Leica LAS X Navigator software. The number of positive cells was counted in three sections per animal and presented as the number of positive cells per high-power field.
5
Astrocytic Activation Analysis by GFAP
6
Histopathological Assessment of Hippocampal Damage
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For histopathological examinations, hippocampal tissue specimens were fixed in buffered neutral formalin (10%). Hippocampal specimens were dehydrated, embedded in paraffin, and sectioned (4–5 µm). Sections were stained using hematoxylin and eosin (H&E) and analyzed using a Nikon Eclipse E200-LED microscope (Tokyo, Japan, 200x and 400× magnification). The degree of hippocampal damage was characterized using a semi-quantitative scoring system, with five grades assigned according to the severity of injury, as follows: 1, minimal injury (<1%); 2, slight injury (1%–25%); 3, moderate injury (26%–50%); 4, moderate/severe injury (51%–75%); and 5, severe injury (76%–100%).47 (link) To detect glial fibrillary acidic protein (GFAP), hippocampal tissue sections were incubated overnight with an anti-GFAP antibody (Santa Cruz, CA, USA) at 4°C, followed by incubation with biotinylated secondary antibody at room temperature for 1-h. The 3,3ʹ-diaminobenzidine (DAB) peroxidase substrate kit was used to develop a brown color. The images were observed at a magnification of 400×, using a Nikon Eclipse E200 LED (Tokyo, Japan).
7
Glioma Cell Line Propagation and Signaling
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U87MG glioma cell line was purchased from the American Tissue Culture Collection (Rockville, MD). These cell lines were propagated in RPMI (Invitrogen, Carlsbad, CA), with 10% FBS (Hyclone) and 1% penicillin/streptomycin as described before [25 (link)]. Antibodies against phospho-AKT (ser473), AKT, PTEN, phospho-ERK (p42/44 MAP kinase; Thr202/Tyr204), ERK (p42/44 MAP kinase) and PCNA were purchased from Cell Signaling Technology (Beverly, MA). Tubulin and anti-CD31 antibodies were procured from BD Biosciences (San Jose, CA). Anti-GFAP antibody, rabbit ImmunoCruz Staining system, mouse ImmunoCruz Staining system and rat ABC staining system were procured from Santa Cruz Biotechnology, Inc, (Santa Cruz, CA). LY294002 and PD98059 were purchased from Calbiochem (San Diego, CA). Vitronectin, β-actin and X-gal were purchased from Sigma-Aldrich, St. Louis, MO. RGDS was procured from Biomol International (Plymouth, PA). HRP-tagged anti-rabbit IgG and anti-mouse IgG were obtained from Amersham Life Sciences (UK). Goat anti-mouse and anti-rabbit IgG (H + L)-AP (human adsorbed) were purchased from Southern Biotechnology, Inc. (Birmingham, Alabama). SF1126 is a vascular targeted pan PI-3 kinase drug developed in collaboration with SignalRx pharmaceuticals.
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