Ab10802

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab10802 is a primary antibody that recognizes the protein target ABC. It is suitable for use in common laboratory techniques such as Western blotting and immunohistochemistry. Further details about the specific applications and performance characteristics of this product are not available.

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10 protocols using ab10802

Immunofluorescence Staining of 8-OHG and NeuN

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For immunofluorescence, 5 μm-thick coronal cryostat sections were rinsed in PBS after dewaxing and rehydration, and then immersed in 0.01 M citrate buffer (pH 6.0) at 95–100°C for 5 min. The treated sections were blocked for 60 min in blocking buffer solution (0.1% Triton^TM X-100/1× PBS 5% normal serum). Anti-8-hydroxyguanosine (goat polyclonal, diluted 1:200, Abcam, Cambridge, UK, ab10802) and anti- NeuN (rabbit monoclonal, diluted 1:300, Abcam, ab177487) were used and processed at 4°C overnight. After washing with PBST, all secondary antibodies were incubated for 1 h at RT in the dark. ThermoFisher Scientific (Waltham, MA, USA) Alexa Fluor 488 donkey anti-goat (Invitrogen) and Alexa Fluor 488 goat anti-rabbit (Invitrogen) were used for the recognition of primary antibodies, respectively. After incubation, the sections were washed with PBST and then stained with DAPI (Beyotime Institute of Biotechnology, Haimen, China) for 5 min to reveal the nuclei. The images were observed with a laser scanning confocal microscope (Leica, Wetzlar, Germany).

Wang D., Yu H., Xu B., Xu H., Zhang Z., Ren X., Yuan J., Liu J., Guo Y., Spencer P.S, & Yang X. (2018). TRPC1 Deletion Causes Striatal Neuronal Cell Apoptosis and Proteomic Alterations in Mice. Frontiers in Aging Neuroscience, 10, 72.

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Histological Analysis of Heart Tissue

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Heart tissues were fixed with 4% paraformaldehyde and imbedded in paraffin; At the left ventricle (LV) papillary muscle level, tissue sections (4-μm thickness) of the hearts were sliced and stained with Masson trichrome, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL, In Situ Cell Death Detection Kit, POD, 11684817910, Roche, Switzerland), and immunohistochemical methods using anti-p53 (OP33, Millipore, United States) and 8-OHdG (ab10802, Abcam, United States). The immunostaining of p53 was performed according to the immunohistochemical protocol from Millipore using p53 antibody (1:100 dilution). Digital photomicrographs were taken at magnification of 200×, and five random fields from each section were selected and analyzed with unawareness of animal groups. Size of fibrosis, apoptosis cardiomyocytes, p53 and 8-OHdG positive cells were measured in five sections from each heart, and the mean value was calculated.

Chi J., Li Z., Hong X., Zhao T., Bie Y., Zhang W., Yang J., Feng Z., Yu Z., Xu Q., Zhao L., Liu W., Gao Y., Yang H., Yang J., Liu J, & Yang W. (2018). Inhalation of Hydrogen Attenuates Progression of Chronic Heart Failure via Suppression of Oxidative Stress and P53 Related to Apoptosis Pathway in Rats. Frontiers in Physiology, 9, 1026.

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Skin Redox Status Evaluation

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Glutathione (GSH) levels were estimated in supernatants from skin homogenates using GSH –glo assay kit (Promega, Madison, WI). Results were normalized to mg tissue for mouse skin and mg protein for human skin.
Carbonyl protein levels were determined using the OxyElisa kit (Millipore, #S7250, Billerica, MA). Homogenized skin protein concentration was determined by staining with coomassie. Oxyelisa plates were coated overnight at 4 °C with skin homogenate supernatants diluted to a protein concentration of 0.025 μg/mL in Lysis Buffer. Oxyelisa assay was performed following the Millipore protocol.
DNA damage was determined by assessing 8h-hydroxyguanosine by immunohistochemistry. Slides were deparrafinized and rehydrated. Antigen retrieval was performed using sodium citrate buffer and 0.1% trypsin according to manufacturer’s protocol (Abcam). Slides were blocked, stained with Polyclonal Goat anti 8-Hydroxyguanosine primary antibody (Abcam, ab10802), stained with Horseradish Peroxidase conjugated Rabbit anti-Goat IgG (Abcam, ab6741) and developed with 3,3′ Diaminobenzidine (Dab) substrate kit (Abcam, ab64238) according to manufacturer’s protocol with the exception of a 1 min incubation with the Dab.

Brand R.M., Epperly M.W., Stottlemyer J.M., Skoda E.M., Gao X., Li S., Huq S., Wipf P., Kagan V.E., Greenberger J.S, & Falo LD J.r. (2016). A Topical Mitochondria-Targeted Redox Cycling Nitroxide Mitigates Oxidative Stress Induced Skin Damage. The Journal of investigative dermatology, 137(3), 576-586.

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Immunohistochemical Analysis of Oxidative Stress Markers

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The paraffin samples were cut at 2-μm thickness, deparaffinized, and rehydrated. After inactivation of endogenous peroxidase with 3% H₂O₂, slides were pre-incubated with 10% fetal calf serum in Dako buffer (Dako) to block non-specific reactions. The samples were incubated with rabbit polyclonal anti TIM-1 (10 μg/ml, ThermoFisher, PA5-20244) and goat polyclonal antibody against 8-hydroxyguanosine (8-OHdG) (2 μg/ml, Abcam, San Francisco, CA, USA, ab10802) overnight at 4°C. Anti-Nox1 (Abcam, San Francisco, CA, USA, ab131088), anti Nox2/gp91phox (Abcam, San Francisco, CA, USA, ab80508), and anti-Nox4 (Abcam, San Francisco, CA, USA, ab133302) were used to detect the various NDAPH homologues. Secondary antibodies were applied for 1 h at room temperature in 10% FCS/Dako buffer. Finally, the sections were incubated with avidin peroxidase (1:100, Sigma Aldrich) in 10% FCS/Dako buffer for 1 h at room temperature. Antibody binding was routinely visualized using DAB (Sigma Aldrich). The sections were counterstained with haematoxylin, dehydrated, and mounted in Eukitt.

Jeong B.Y., Lee H.Y., Park C.G., Kang J., Yu S.L., Choi D.R., Han S.Y., Park M.H., Cho S., Lee S.Y., Hwang W.M., Yun S.R., Ryu H.M., Oh E.J., Park S.H., Kim Y.L, & Yoon S.H. (2018). Oxidative stress caused by activation of NADPH oxidase 4 promotes contrast-induced acute kidney injury. PLoS ONE, 13(1), e0191034.

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Comprehensive Bone and Liver Analysis

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Livers were fixed in 10% zinc formalin, processed for paraffin sectioning (5-μm sections) and stained with F4/80 (abcam, Cat# ab6640, 1:100) and Iba1 (ThermoFisher, Cat# PA5-27436, 1:100). Femora were fixed in 10% zinc formalin and decalcified using 10% EDTA. Decalcified bones were then processed for paraffin sectioning (5-μm sections) and stained with hematoxylin and eosin. Tibiae sections from 52 weeks old mice were stained with anti-8-Hydroxydeoxyguanosine (abcam, Cat#ab10802, 1:200). Tartrate resistant acid phosphatase (TRAP) staining of bone sections was done using a kit according to the manufacturer’s instructions (Sigma 387A). Number of osteoclasts on trabecular bone surface was counted manually.

Liu Z., Han T., Werner H., Rosen C.J., Schaffler M.B, & Yakar S. (2017). Reduced serum IGF-1 associated with hepatic osteodystrophy is a main determinant of low cortical but not trabecular bone mass. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 33(1), 123-136.

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Quantification of DNA Damage and Antioxidant Levels

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DNA damage was determined by staining sections for 8-oxo and CPD by immunohistochemistry using Polyclonal Goat anti 8-Hydroxyguanosine primary antibody (Abcam, ab10802) and Anti-Thymine Dimer mAb (clone KTM53) and quantitated as described earlier.
Glutathione (GSH) levels in supernatant from skin homogenates were measured using GSH–glo assay kits. (Promega, Madison, WI). For each skin sample, a 6 mm punch biopsy was homogenized in 300 ul of PBS+2mM EDTA and centrifuged at 1000g for 10 minutes and assayed following manufacturer’s instructions [24 ].

Brand R.M., Stottlemyer J.M., Paglia M.C., Carey C.D, & Falo LD J.r. (2020). Ethanol Consumption Synergistically Increases Ultraviolet Radiation Induced Skin Damage and Immune Dysfunction.. Journal of dermatological science, 101(1), 40-48.

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Quantifying Oxidative Nucleic Acid Damage

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Oxidative nucleic acid damage was assessed by 8-OHdG immunofluorescence in the aorta sections. After deparaffinization and hydration, sections were heat treated (1 mmol/l EDTA, pH 8.0) for 15 min in a microwave. Primary 8-hydroxyguanosine antibody incubation (8-OHG, 1:200, Ab10802; Abcam, Cambridge, UK) was performed overnight at 4°C, followed by rinsing in TBS+ 0.05% Tween20, and incubation with secondary antibody (Alexa-fluor-488-conjugated, 1:300, A-11055; Molecular Probes, Eugene, Oregon, USA). Lipofuscin-mediated autofluorescence was removed by using 0.1% Sudan Black B (Sigma, St Louis, Missouri, USA). Slides were mounted using ProLong Gold Anti-fade mounting media containing DAPI (Molecular Probes) for nuclei staining. All sections were analysed in Live Cell Microscope (Zeiss, Cambridge, UK), using fluorescent filters (Alexa-fluor-488 and DAPI), and obtained images for each fluorescent filter. 8-OHG analysis was performed by measuring the intensity of immunofluorescence in Alexa-fluor-488 [CTTF = Integrated Density – (Area of selected tissue X Mean fluorescence of background readings].

Alves-Lopes R., Lacchini S., Neves K.B., Harvey A., Montezano A.C, & Touyz R.M. (2023). Vasoprotective effects of NOX4 are mediated via polymerase and transient receptor potential melastatin 2 cation channels in endothelial cells. Journal of Hypertension, 41(9), 1389-1400.

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Immunoblotting Antibody Validation

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The anti-p65 antibody (sc-372), the anti-PDGFR-β antibody (sc-432), the anti-β-actin antibody (sc-517582) and the anti-NG2 antibody (sc-166251) were obtained from Santa Cruz Inc. The antibodies anti-CD54 (ICAM-1) (555511), anti-CD62E (E-selectin) (551145), anti-CD106 (VCAM-1) (555647) and IgG1-κ Isotype Control (555749) were purchased from BD Biosciences (Heidelberg, Germany). Peroxidase-labeled anti-rabbit antibody (NIF 824) and peroxidase-labeled anti-mouse antibody (NIF 825) were obtained from GE Healthcare (Freiburg, Germany). The anti-HO-1 antibody (ADI-SPA-895) was from Enzo Life Sciences (Lörrach, Germany). The anti-α-tubulin antibody (ab56676), the anti-myeloperoxidase (MPO) antibody (ab9535), the anti-ICAM-1 antibody (ab124760), the anti-8-OHDG antibody (ab10802), the anti-eNOS antibody (ab5589), the anti-VCAM-1 antibody (ab134047), the anti-CD68 antibody (ab1252212) and the secondary biotinylated goat anti-rabbit antibody (ab64256) were purchased from Abcam.

Ampofo E., Berg J.J., Menger M.D, & Laschke M.W. (2019). Maslinic acid alleviates ischemia/reperfusion-induced inflammation by downregulation of NFκB-mediated adhesion molecule expression. Scientific Reports, 9, 6119.

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Oxidative DNA Damage in Neutrophils

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Neutrophils grown on coverslips in the lower chamber were fixed with 4% PFA overnight, permeabilized in 0.5% Triton X‐100 in phosphate‐buffered saline (PBS) for 30 minutes, and incubated in 1× blocking buffer (5% bovine serum albumin in PBS) for 30 minutes. Oxidized DNA was detected with a biotinylated anti‐8‐OHG antibody (ab10802; Abcam) used at 1 μg/mL, and mitochondria (TOMM20) were detected using rabbit mAb EPR15581 (1:250, ab186734; Abcam), followed by incubation with AlexaFluor 488‐conjugated and AlexaFluor 647‐conjugated donkey anti‐goat and rabbit‐conjugated antibodies at 1:200 (Abcam), and DAPI was used to stain the DNA. Images were acquired using a confocal microscope (Zeiss LSM 510 Meta).

Liu L., Mao Y., Xu B., Zhang X., Fang C., Ma Y., Men K., Qi X., Yi T., Wei Y, & Wei X. (2019). Induction of neutrophil extracellular traps during tissue injury: Involvement of STING and Toll‐like receptor 9 pathways. Cell Proliferation, 52(3), e12579.

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Quantifying Amyloid-beta and Oxidative DNA Damage

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Brain tissues were fixed for 48 h in 4% paraformaldehyde at 4°C, dehydrated in ethanol and embedded in paraffin. Coronal sections of the hippocampus were obtained to analyze Aβ_1–42 protein and DNA oxidative damage in the form of 8-hydroxydeoxyguanosine (8-OHdG) DNA adducts, After dewaxing and rehydration, the sections were treated with 0.01 M citrate buffer (pH 6.0) and 0.1% Tween-20 at 90–95°C for 5 min for antigen retrieval. Tissues were then incubated at 4°C overnight with the primary antibody to 8-OHdG (1:200, Abcam, ab10802) and to Aβ_1–42 (1:100, Abcam, ab10148). After washing with PBS, tissues were stained for 1 h in darkness at RT with the secondary antibody, namely FITC-goat anti-rabbit IgG (H + L), and then counterstained with DAPI (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) for 1 min to reveal the nuclei, Tissues were examined and images taken with a laser scanning confocal microscope (Leica, Wetzlar, Germany).

Yu H., Lin X., Wang D., Zhang Z., Guo Y., Ren X., Xu B., Yuan J., Liu J., Spencer P.S., Wang J.Z, & Yang X. (2018). Mitochondrial Molecular Abnormalities Revealed by Proteomic Analysis of Hippocampal Organelles of Mice Triple Transgenic for Alzheimer Disease. Frontiers in Molecular Neuroscience, 11, 74.

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