The tissue sections were deparaffinized and rehydrated using standard techniques. The sections were incubated with 3% H2O2 for 10 min at room temperature (RT). They were then washed with 1X TBST buffer for 10 min at RT. Primary antibodies were diluted in 2.5% normal horse serum (Vector Laboratory, Bulingame, CA, USA). The primary antibodies were AGE (TransGenic, Kyoto, Japan), the receptor for AGE (RAGE, Santa Cruz, CA, USA), high mobility group box 1 (HMGB1, Abcam, Boston, MA, USA), 8-hydroxydeoxyguanosine (8-OHdG, Abcam, Boston, MA, USA), and aquaporin5 (AQP5, Bioworld Technology, St. Louis Park, MN, USA). Sections were visualized with the VECTASTAIN Elite ABC Universal Kit (Vector Laboratory, Bulingame, CA, USA). The staining was observed using a BX51 light microscope (Olympus, Tokyo, Japan). Ten unique fields of view were randomly selected in each slice at 100× magnification. Image-Pro software (Media Cybernetics, Rockville, MD, USA) was used for semi-quantitative analysis of the intensity of the immunohistochemical positive signal. The average optical density per unit area (mm2) was calculated for the relative amount of protein.
8 hydroxydeoxyguanosine 8 ohdg
Manufactured by Abcam
Sourced in United States
8-hydroxydeoxyguanosine (8-OHdG) is a modified nucleoside that is formed when DNA is damaged by reactive oxygen species. It is a commonly used biomarker for oxidative stress.
Automatically generated - may contain errors
Lab products found in correlation
Phosphotungstic acid, by Merck Group (2 mentions)
2 thiobarbituric acid, by Merck Group (2 mentions)
Bicinchoninic protein determination assay, by Merck Group (2 mentions)
Rabbit polyclonal anti prdx1, by Abcam (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Lumi light, by Roche (2 mentions)
Donkey anti rabbit igg, by Cedarlane (2 mentions)
Goat anti mouse igg, by Cedarlane (2 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
Vectastain universal elite abc kit, by Vector Laboratories (1 mentions)
Hmgb1, by Abcam (1 mentions)
Image pro software, by Media Cybernetics (1 mentions)
Bx51 light microscope, by Olympus (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
Biotinylated horse anti mouse antibody, by Vector Laboratories (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Anti 8 ohdg antibody, by StressMarq Biosciences (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Proteinase k, by Merck Group (1 mentions)
8 protocols using 8 hydroxydeoxyguanosine 8 ohdg
1
Immunohistochemical Analysis of Aging Markers
2
Oxidative Stress Protein and DNA Assays
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tert-butyl hydroperoxide (tert-BHP), SDS, phosphotungstic acid, buthylated hydroxytoluene, 2-thiobarbituric acid and malonaldehyde bis(dimethyl acetal), the Bicinchoninic protein determination assay and the anti-α-tubulin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The following were purchased from Abcam Inc., (Cambridge, MA, USA): rabbit polyclonal anti-PRDX1, monoclonal anti-PRDX4, monoclonal anti-PRDX6, the antigenic peptide used to raise the anti-PRDX1 antibody and 8-hydroxy-deoxyguanosine (8-OHdG). The anti-8-OHdG antibody was purchased from StressMarq Biosciences Inc., (Victoria, BC, Canada). Biotinylated horse anti-mouse antibody and Horse Serum were purchased from Vector Labs. Alexa-555 fluor streptavidin (1 mg ml−1 in H2O) and ProLong Gold antifade with DAPI were purchased from Invitrogen Life Technologies (Burlington, ON, Canada). Nitrocellulose (0.22 μm pore size; Osmonics Inc., MN, USA), donkey anti-rabbit IgG and goat anti-mouse IgG, both conjugated to horseradish peroxidase (Cedarlane Laboratories Ltd., Hornby, ON, Canada), an enhanced chemiluminescence kit (Lumi-Light; Roche Molecular Biochemicals, Laval, QC, Canada) and radiographic films (Fuji, Minamiashigara, Japan) were also used for immunodetection of blotted proteins. Other chemicals used were of at least reagent grade.
3
Immunohistochemical Analysis of Oxidative Stress and Inflammation
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The paraffin-embedded blocks were sectioned for immunohistochemistry analysis. Sections were treated with 20 mg/L proteinase K (dilution: 1∶1000; Sigma-Aldrich, USA) at 37°C for 30 min for antigen retrieval. After blocking the endogenous peroxidase activity with 0.3% hydrogen peroxidase for 15 min, the sections were treated with 8-hydroxydeoxyguanosine (8-OHdG; 1∶8000; Abcam, USA) or NF-κB (Cell Signaling Technology, 1∶100) primary antibody at 4°C overnight. The slides were incubated in biotinylated secondary IgG antibodies at 37°C for 30 min, and then visualized using DAB for 2–5 min. Mayer’s hematoxylin was used to counterstain the sections, which were then dehydrated and mounted. For negative control, PBS was used in place of primary antibodies. The cells with positive staining were determined by counting the percentage of stained cells using the Image Pro Plus 7.0 analyzer. A minimum of 1,000 cells were counted for each tumor specimen.
4
Quantitative Pancreatic Tissue Analysis
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Pancreatic tissue was fixed in 10% formalin overnight and then embedded in paraffin. Ten 4‐μm sections were cut from each tissue block (n = 5 for each group). For immunohistochemistry, the tissue sections were blocked with peroxidase blocking agent for 5 min and washed with TBST, then blocked with protein blocking serum‐free buffer (DAKO, Carpinteria, CA) for 5 min. Sections were incubated in primary antibodies against insulin (Santa Cruz Biotechnology, Dallas, TX), glucagon (Bioworld, St. Louis Park, MN), NOX‐4 (Abcam, Cambridge, MA), or 8‐hydroxydeoxyguanosine (8‐OHdG; Abcam) for 2 h at room temperature. After washing with TBST, samples were then incubated in secondary antibodies (DAKO) at room temperature for 30 min, then the chromogen was added. All sections were H&E stained and observed at 200× magnification with a light microscope (Nikon, Tokyo, Japan). Islet areas and entire section areas were measured using iSolution DT 36 software (Carl Zeiss, Oberkochen, Germany) (Ka et al. 2015 ).
5
Liver Inflammation and Oxidative Stress Biomarkers
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After determining the protein concentration of 200-mg frozen liver samples, levels of TNF-α (Biosource International, Camarillo, CA), IL-6 (Quantikine HS; R&D Systems, Minneapolis, MN), CCL2 (LifeSpan Biosciences, Seattle, WA, USA), malondialdehyde (MDA, NWLSS, Vancouver, WA, USA), and 8-hydroxydeoxyguanosine (8-OHdG, AbCam, Cambridge, UK) were determined using enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions.
6
Immunofluorescence Staining of Neural Markers
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Immunofluorescence staining was performed as we described in detail before [17 (link)]. In brief, the sections were first fixed by 0.1% Triton X-100 and then were blocked by 5% BSA for 2 h. After three washes with PBS, brain tissues or cultured neurons were incubated with primary antibodies against Iba-1 (1: 50, Santa Cruz Biotechnology or 1 : 100, Abcam), myeloperoxidase (MPO, 1 : 50, Santa Cruz Biotechnology), 8-hydroxydeoxyguanosine (8-OHdG) (1 : 100, Abcam), Nrf2 (1 : 100, Abcam), caspase-1 p20 (1 : 50, Santa Cruz Biotechnology), and NeuN (1 : 200, EMD Millipore). Sections were then incubated with corresponding secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594) overnight at 4°C. Fluorescence microscopy imaging was examined under a ZEISS HB050 inverted microscope system. The fluorescently stained cells were recorded using Image J program.
7
Oxidative Stress Biochemical Assay
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Tert-butyl hydroperoxide (t-BHP), sodium dodecyl sulfate (SDS), phosphotungstic acid, buthylated hydroxytoluene, 2-thiobarbituric acid and malonaldehyde bis(dimethyl acetal), the Bicinchoninic protein determination assay and the anti-α-tubulin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The following were purchased from Abcam Inc., (Cambridge, MA, USA): rabbit polyclonal anti-PRDX1, mouse monoclonal anti-PRDX6, mouse monoclonal anti-4-Hydroxynonenal (4-HNE), rabbit polyclonal anti-catalase, and 8-hydroxy-deoxyguanosine (8-OHdG). Anti-thioredoxin 1 antibody was purchased from Cell Singaling Thecnology (Danvers, MA, USA), Polyvinylidene fluoride (PVDF) membranes (0.22 µm pore size; Osmonics Inc., Minnetonka, MN, USA), donkey anti-rabbit IgG and goat anti-mouse IgG, both conjugated to horseradish peroxidase (Cedarlane Laboratories Ltd., Hornby, ON, Canada), an enhanced chemiluminescence kit (Lumi-Light; Roche Molecular Biochemicals, Laval, QC, Canada) and radiographic films (Denville Scientific, Inc., Saint-Laurent, QC, Canada) were also used for immunodetection of blotted proteins. Other chemicals were used at the reagent level.
8
Molecular Mechanisms of SIRT1 in Fibrosis
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Antibodies and other reagents SRT1720 was purchased from BioChemPartner (Shanghai, China). Antibodies against SIRT1, HIF1A, GLUT1, fibronectin, collagen IV, E-cadherin, α-SMA, NF-KB p65 and 8-hydroxydeoxyguanosine (8-OHdG) were obtained from Abcam. Antibodies for transforming growth factor beta 1 (TGFB1), NAD(P)H oxidase 4 (NOX4), CTGF, SNAIL, Histone H3 and β-actin were purchased from Proteintech (Chicago, USA). Goat anti-rabbit or mouse IgG-HRP secondary antibodies, TRITC-labelled goat anti-rabbit secondary antibody and FITC-labelled goat anti-rabbit or mouse IgG secondary antibodies were purchased from Zhongshan Goldenbridge Biotechnology (Beijing, China). Biochemical parameters in urine and plasma were measured with the reagent kits purchased from BioSino Biotechnology and Science (Beijing, China). PVDF membranes were purchased from Millipore. All culture media were from Gibco-BRL, and the Lipofectamine RNAiMAX was purchased from Invitrogen Life Technologies.
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