Ab4674

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab4674 is a laboratory equipment product offered by Abcam. It is a core component designed for specific research and experimental applications. The detailed technical specifications and intended uses of this product are not included in this response to maintain an unbiased and factual approach.

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Lab products found in correlation

Ab177487, by Abcam (6 mentions) Ab5392, by Abcam (5 mentions) Ab178846, by Abcam (5 mentions) Ab5076, by Abcam (4 mentions) Ab222782, by Abcam (4 mentions) Ab18102, by Abcam (4 mentions) Ab7751, by Abcam (4 mentions) Ab104225, by Abcam (4 mentions) A11039, by Thermo Fisher Scientific (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions) Ab200999, by Abcam (3 mentions) Ab78078, by Abcam (3 mentions) Alexa fluor 488, by Thermo Fisher Scientific (3 mentions) Hoechst 33342, by Thermo Fisher Scientific (3 mentions) Ab48004, by Abcam (2 mentions) Ctb alexa 555, by BrainVTA (2 mentions) Anti p70s6k, by Cell Signaling Technology (2 mentions) Anti p p70s6k, by Cell Signaling Technology (2 mentions) Anti β actin, by Santa Cruz Biotechnology (2 mentions) Ab14692, by Abcam (2 mentions)

Close spelling variants of this product

Chicken anti-GFAP (ab4674, (2 citations)

95 protocols using ab4674

Immunofluorescence Staining of Neural Stem Cells

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SFME cells were cultivated on fibronectin-coated glass coverslips. Subsequently, cells were differentiation induced as described above. Both induced cells and untreated controls were methanol fixed and treated with 0.2% Tween-20 for 2 minutes. Coverslips were blocked with goat serum and incubated for 1 h with chicken antibody polyclonal to GFAP (ab4674, Abcam) and rabbit antibody polyclonal to nestin (ab27952, Abcam). Detection was done with an Alexa-488 coupled secondary antibody against chicken and Alex-594 coupled secondary antibody against rabbit.
Differentiating neural stem and progenitor cells (primary mesencephalon E14 neurosphere cells) grown on coated glass slides were fixed in ice-cold methanol for 20 minutes. Slides were washed in PBS for 5 minutes and treated with 0.2% Tween-20 for 2 minutes. Subsequently, slides were blocked with goat serum and incubated for 1 h with chicken antibody polyclonal to GFAP (ab4674, Abcam) and rabbit antibody polyclonal to nestin. Detection was done with an Alexa-488 coupled secondary antibody against chicken and Alex-594 coupled secondary antibody against rabbit.

Fischer U., Backes C., Raslan A., Keller A., Meier C, & Meese E. (2015). Gene amplification during differentiation of mammalian neural stem cells in vitro and in vivo. Oncotarget, 6(9), 7023-7039.

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Immunostaining of Stem Cell and Neuronal Markers

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Mounted sections were incubated for 1 h at room temperature with and blocked using blocking buffer containing (Sigma‐Aldrich, #G9023) and 0.1% (v/v) Triton X100 (Sigma‐Aldrich, #9036‐19‐5), and then incubated with primary antibodies diluted in blocking solution overnight at 4 °C. The following primary antibodies were used for immunostaining: Nanog (rabbit, 1:100; Abcam, #ab80892), OCT4 (rabbit, 1:100; Abcam, #ab19857), SSEA4 (mouse, 1:200; Abcam, #ab16287,), NeuN (rabbit, 1:500; Cell Signaling, #24307S), MAP2 (rabbit, 1:1000; Abcam, #ab5392), β‐tubulin III (TUJ1) (mouse, 1:1000; Abcam, #ab78078), Synaptophysin (mouse, 1:500; Proteintech, #17785‐1‐AP), PSD‐95 (rabbit, 1:500; Proteintech, #20665‐1‐AP), SOX2 (rabbit, 1:100; Abcam, #ab97959), NDUFB10 (rabbit, 1:350; Abcam 196 019), TFAM (mouse, 1:1000; Abcam #ab119684), TOMM20 (mouse, 1:350; Abcam, #ab56783), GFAP (chicken, 1:500; Abcam #ab4674), DCX (mouse, 1:200; Abcam #ab135349), SATB2 (rabbit, 1:400; Abcam #ab4674), CTIP2 (rat, 1:500; Abcam, #ab18465), OLIG2 (rabbit, 1:500; Abcam, #ab42453). Alexa Fluor Dyes (Invitrogen, #A11008, #A21449, #A11012, #A21141, #A11042, #A21236) were used at 1:800 dilution as secondary antibodies. Slides were mounted using ProLong diamond antifade mounting medium (SouthernBiotech, #0100‐20), and analyzed using the Leica TCS SP8 STED 3X (Leica microsystems).

Chen A., Yangzom T., Hong Y., Lundberg B.C., Sullivan G.J., Tzoulis C., Bindoff L.A, & Liang K.X. (2024). Hallmark Molecular and Pathological Features of POLG Disease are Recapitulated in Cerebral Organoids. Advanced Science, 11(18), 2307136.

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Antioxidant Compound Characterization

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Resveratrol and xanthohumol were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Trolox and Mn-TM-2-PyP were obtained from Millipore Sigma (St. Louis, MO, USA). For experiments, Resveratrol and xanthohumol were dissolved in dimethyl sulfoxide (Millipore Sigma, St. Louis, MO, USA), Mn-TM-2-PyP was dissolved in 0.1 M phosphate-buffered saline pH 7.4 (PBS) and Trolox was dissolved in ethanol.
The following antibodies were used for immunoblotting experiments: chicken anti-glial fibrillary acid protein (GFAP; ab4674; AbCam, Cambridge, MA, USA; 1:5000 dilution); rabbit anti-Nox4 (ab133303; AbCam; 1:5000 dilution); mouse anti-Nrf2 (VMA00224; Biorad Laboratories, Hercules, CA; 1:1000 dilution); rabbit anti-Sod2 (A1340; ABclonal, Woburn, MA; 1:2000 dilution). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control (rabbit anti-GAPDH; sc-25778; Santa Cruz Biotechnology, Dallas, TX; 1:2000 dilution). Secondary antibodies were horseradish peroxidase-conjugated and obtained from GE Healthcare (Chicago, IL, USA).
Immunocytochemistry was performed using chicken anti-glial fibrillary acid protein (GFAP; ab4674; AbCam; 1:1000 dilution) and Alexa Fluor^® 594-labeled goat anti-chicken secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Ghosh A.K., Rao V.R., Wisniewski V.J., Zigrossi A.D., Floss J., Koulen P., Stubbs EB J.r, & Kaja S. (2020). Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants. Antioxidants, 9(4), 324.

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Immunofluorescence Staining of Brain Sections

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For immunofluorescence staining, brain sections of six animals per genotype were utilized. Remaining cryoprotection solution was removed by rinsing with PBS. For improvement of antibody penetration, sections were incubated in 0.3 M glycine, 0.2% Triton-X100, and 10% DMSO in dH₂O at room temperature (RT) for 1 h. Furthermore, unspecific antibody binding was blocked using 0.3% donkey serum, 0.2% Triton-X100 in PBS at 37 °C for 1 h. For astrocyte labeling, primary antibodies were incubated at 4 °C for 72 h: GFAP (Abcam Cat# ab4674, RRID:AB_304558, 1: 1.000), ALDH1L1 (Abcam Cat# ab87117, RRID:AB_10712968, 1:500), and S100β, (Abcam Cat# ab52642, RRID:AB_882426, 1:200). Forebrain sections were rinsed using PBS and subsequently incubated with secondary antibodies at RT for 1 h: anti-chicken Alexa 488 (Jackson ImmunoResearch Labs Cat# 703-545-155, RRID:AB_2340375, 1:500) and anti-rabbit Alexa 647 (Jackson ImmunoResearch Labs Cat# 711-605-152, RRID:AB_2492288, 1:500). Secondary antibody was rinsed and nuclei were counterstained using DAPI. Imaging was performed using the Axio Observer fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were processed and analyzed applying ImageJ and Fiji software [37 (link), 38 (link)].

Schneider Y., Gauer C., Andert M., Hoffmann A., Riemenschneider M.J., Krebs W., Chalmers N., Lötzsch C., Naumann U.J., Xiang W., Rothhammer V., Beckervordersandforth R., Schlachetzki J.C, & Winkler J. (2024). Distinct forebrain regions define a dichotomous astrocytic profile in multiple system atrophy. Acta Neuropathologica Communications, 12, 1.

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Microglia and Astrocyte Immunostaining Protocol

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The mice were anesthetized with 400 mg/kg chloral hydrate (Sigma-Aldrich, USA), then 0.9% saline was infused into the hearts of the mice, and their brains were dissected and fixed in 4% paraformaldehyde phosphate buffer at 4 °C for 48 hours, and then cryopreserved with 30% sucrose. A cryostat was used to cut 30 µm continuous sections. Next, the sections were incubated with 0.3% hydrogen peroxide in methanol for 10 minutes, and then blocked with 10% normal goat serum in PBS for 20 minutes. Single staining of the IBA1 (ionized calcium binding adaptor molecule 1) and GFAP (glial fibrillary acidic protein), was achieved by blocking with anti-Iba1 (1:200, goat polyclonal anti-Iba1, Abcam, USA, #ab5076) and anti-GFAP (1:2,500, chicken polyclonal ant-GFAP, Abcam, USA, #ab4674) and incubated at 4 °C overnight in the solution. After washing three times with PBS the next day, the sections were incubated with an Alexa 484-conjugated IgG secondary antibody (Invitrogen, CA, USA) for 2 hours, and finally 4’,6-diamino-2-phenylindole (DAPI) (Solarbio, China) was used for the fluorescent nuclear staining. After 15 minutes of incubation, the coverslip was thoroughly washed with distilled water.

Li N., Zhang X., Gu Z., Su C, & Lian H. (2021). Young plasma attenuates cognitive impairment and the cortical hemorrhage area in cerebral amyloid angiopathy model mice. Annals of Translational Medicine, 9(2), 147.

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Immunofluorescence Staining of Stem Cell Markers

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Immunofluorescence was performed as described previously [56 (link)]. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, and incubated with primary antibody overnight at 4 °C, followed by FITC- or rhodamine-conjugated secondary antibody. The primary antibodies against PLOD1 (Sigma‐Aldrich), CD133 (1:1000, #ab222782, Abcam), CD44 (Abcam), nestin+ (1:500, #ab18102, Abcam), GFAP (1:1000, #ab4674, Abcam), and β III tubulin (1:1000, #ab7751, Abcam) were the same as described above. DAPI (Sigma‐Aldrich) was used to stain the nuclei. A laser scanning confocal microscope was used to detect and photograph immunofluorescence expression (Olympus, Tokyo, Japan).

Wang Z., Shi Y., Ying C., Jiang Y, & Hu J. (2021). Hypoxia-induced PLOD1 overexpression contributes to the malignant phenotype of glioblastoma via NF-κB signaling. Oncogene, 40(8), 1458-1475.

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Investigating Synaptic Regulation in Alzheimer's

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Retinoic acid was obtained from Sigma-Aldrich China (Shanghai, China; R2625). Rapamycin was purchased from MedChemExpress (Shanghai, China; HY-10219). CTB-Alexa 488 and CTB-Alexa 555 were bought from BrainVTA (Wuhan, Hubei, China). The following antibodies were used in western blot: anti-CD82 (ab66400 & ab135779; Abcam; 1:1000); anti-synaptophysin (ab14692; Abcam; 1:1000); anti-p70S6k (2708T; Cell Signaling Technology; 1:1000); anti-p-p70S6k(9234T;Cell Signaling Technology; 1:1000); anti-β-actin (sc-47778; Santa Cruz; 1:1000); anti-TRAF2 (ab126758; Abcam; 1:1000); anti-Raptor (20984-1-AP; Proteintech; 1:1000).The following antibodies were used in immunofluorescence: anti-CD82 (ab66400; Abcam; 1:200); anti-Tuj1 (801201; Biolegend; 1:1000); anti-Iba1(ab48004;Abcam;1:100); anti-GAFP(ab4674; Abcam;1:100); anti-mCherry (ab167453; Abcam; 1:100); anti-synaptophysin (ab14692; Abcam; 1:200); anti-pS6(Ser 240/244)(5364T; Cell Signaling Technology; 1:1000); anti-TRAF2 (ab126758; Abcam; 1:400); anti-Raptor (20984-1-AP; Proteintech; 1:1000); anti-Lamp1 (15665, Cell Signaling Technology; 1:500).
Anti-β-Amyloid (2450; Cell Signaling Technology; 1:100) was used in immunohistochemistry.

Ye M., Huang J., Mou Q., Luo J., Hu Y., Lou X., Yao K., Zhao B., Duan Q., Li X., Zhang H, & Zhao Y. (2021). CD82 protects against glaucomatous axonal transport deficits via mTORC1 activation in mice. Cell Death & Disease, 12(12), 1149.

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Immunofluorescent Detection of GFP-Labeled BMSC

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To detect GFP-labeled BMSC, paraffin-embedded sections were twice deparaffinized with xylene for 5 minutes, and rehydrated in a series of graded alcohol solutions (70%–100%). Endogenous peroxidases were inhibited by incubation with 3% H2O2 in PBS buffer. For antigen retrieval, the samples were heated to 98°C–99°C in antigen-retrieval buffer (10 mMTri-sodium citrate, 0.05% Tween 20, pH 6.0) and incubated for 30 minutes in the pressurized vessel. Nonspecific staining was blocked with a mixture of sera in 1.5%PBS for 30 minutes at room temperature, and incubated in the mixture of two primary antibodies in a pair-wise fashion with the mouse monoclonal anti-GFP antibody (SC-9996) at 1:50 dilutions for 1 hour at room temperature. Following incubation with the appropriate fluorescent-conjugated secondary antibodies, the sections were covered with mounting medium containing DAPI (Santa Cruz, Heidelberg, Germany). The cells were investigated under fluorescence microscope (Leica DMI 4000B; Leica Mycrosystems, Wetzlar, Germany). For other immunostainings, the following antibodies were supplied from Abcam (Cambridge, MA, USA): GFAP (ab4674), anti-vimentin antibody (ab8979) BRN3A (ab81213), and rhodopsin (ab3267). The dilution rate of all primary antibodies was 1:100.

Çerman E., Akkoç T., Eraslan M., Şahin Ö., Özkara S., Vardar Aker F., Subaşı C., Karaöz E, & Akkoç T. (2016). Retinal Electrophysiological Effects of Intravitreal Bone Marrow Derived Mesenchymal Stem Cells in Streptozotocin Induced Diabetic Rats. PLoS ONE, 11(6), e0156495.

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Immunohistochemical Analysis of Neuroinflammation

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Animals were anesthetized with a ketamine cocktail (60 mg/kg, i.p.) and perfused with NS and 4% paraformaldehyde. The brains were then cut into 15 or 30 μm coronal sections using a freezing microtome (Leica Biosystems, Germany) and stored in a cryoprotectant solution consisting of 30% sucrose, 1% polyvinylpyrrolidone (Sigma-Aldrich), and 30% ethylene glycol (Thermo Fisher Scientific, USA) in PBS at -20 °C. Fluorescence immunohistochemistry was performed to detect microglia, astrocytes, and complement C3. Sections were washed with PBS and blocked with 5% normal goat serum (Vector Laboratories, USA) and then incubated with primary antibodies at the following dilutions: Iba-1 (019–19,747; 1:300; Wako Chemicals, USA), GFAP (ab4674; 1:400; Abcam, U.K.), and complement C3 (ab200999; 1:400; Abcam, U.K.). After the primary immunoreaction, the sections were incubated with secondary antibodies conjugated with Alexa Fluor 633 (A20991; 1:200; Thermo Fisher Scientific) or Alexa Fluor 488 (A11001; 1:400; Thermo Fisher Scientific). The staining intensity of the sections was visualized using an M2 microscope (Carl Zeiss, Germany).

Park J.Y., Park J., Baek J., Chang J.W., Kim Y.G, & Chang W.S. (2023). Long-term results on the suppression of secondary brain injury by early administered low-dose baclofen in a traumatic brain injury mouse model. Scientific Reports, 13, 18563.

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Immunolabeling of Neural Markers

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Primary antibodies used were rabbit polyclonal anti-p16^INK4a N-terminal (1:1000) (ab189034; Abcam, Cambridge, UK), chicken polyclonal anti-GFAP (1:2000) (ab4674; Abcam, Cambridge, UK), chicken polyclonal anti-vimentin (1:1000) (ab24525; Abcam, Cambridge, UK), rabbit polyclonal anti-PAX2 (1:200) (71–6000; Invitrogen, Inchinnan, Scotland, UK), and goat polyclonal anti-PAX2 (1:1000) (AF3364; R&D systems, Abingdon, UK).
Secondary antibodies used were (all Alexa Fluor conjugated antibodies from ThermoFisher Scientific, Inchinnan, Scotland, UK) goat antirabbit F(ab’)2 cross-absorbed secondary fluor plus 555 (1:1000) (A48283), goat anti-rabbit 568 (1:1000) (A11036), goat anti-chicken 647 (1:1000) (A21449), donkey anti-goat 647 (1:1000) (A21447), or donkey anti-goat 568 (1:1000) (A11057).

Martinez-Fernandez de la Camara C., Storm T., Salman A., Burgoyne T., Rasmussen M.Q., Orlans H.O., Russell A.J., Davies S.G., Barnard A.R, & MacLaren R.E. (2023). Developmental Expression of the Cell Cycle Regulator p16INK4a in Retinal Glial Cells: A Novel Marker for Immature Ocular Astrocytes?. Journal of Histochemistry and Cytochemistry, 71(6), 301-320.

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