The paraffin-embedded blocks were sliced to sections (4 μm-thick), placed in a 30% hydrogen peroxide solution for 5 min, soaked in 0.01 mol/L citric acid buffer solution (pH = 6.0) after washing in water and heated in a microwave oven for antigen retrieval. Then, the sections were put into a wet box, drops of 5% BSA blocking buffer were added and placed at room temperature for 20 min. After excess liquid removal, acrylamide (ACR) (Abcam, USA) and manganese superoxide dismutase (MnSOD) (Abcam, USA) antibodies were diluted at 1:500 and added dropwise and the sections (in the wet box) placed in a refrigerator at 4°C overnight, washed 3 times with phosphate-buffered saline (PBS), added with drops of secondary antibodies, incubated at 37°C for 30 min, washed again 3 times with PBS and color developed using a DAB kit. Finally, the sections were mounted in neutral balsam and photographed under the light microscope.
Mnsod
Manufactured by Abcam
Sourced in United States, United Kingdom
MnSOD is a mitochondrial superoxide dismutase enzyme that catalyzes the dismutation of superoxide radicals into oxygen and hydrogen peroxide.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (5 mentions)
Cuznsod, by Abcam (4 mentions)
Tgf β1, by R&D Systems (3 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
E cadherin, by BD (2 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions)
Pgc 1α, by Abcam (2 mentions)
Catalase, by Abcam (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
P akt, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
β actin, by Abcam (2 mentions)
Glp 1r, by Abcam (2 mentions)
Active caspase 3, by Merck Group (2 mentions)
Quantity one version 4, by Bio-Rad (1 mentions)
P8340, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Ab48506, by Abcam (1 mentions)
Ecsod, by R&D Systems (1 mentions)
Cytochrome c cyt c, by Cell Signaling Technology (1 mentions)
23 protocols using mnsod
1
Immunohistochemical Analysis of ACR and MnSOD
2
Renal Cortex Protein Analysis
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From tissue lysates from the renal cortex, e-cadherin (BD Biosciences, San Jose, CA, USA), TGFβ-1 (R&D Systems, Minneapolis, MN, USA), MnSOD (Abcam, Cambridge, UK), and β-actin (Sigma) were detected by incubating for 12 h with specific antibodies [15 (link)]. Image analyzer (Quantity One version 4.4.0; Bio-Rad, Hercules, CA, USA) were used to analyze the immunoblot.
3
Whole Cell Lysate Preparation and Protein Analysis
4
Oxidative Stress Signaling in Muscle Atrophy
5
Immunoblotting Analysis of Signaling Proteins
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Immunoblotting analyses were performed as previously described [16 (link)]. Interleukin-17 (IL-17, Cell signaling Technology, Danvers, MA, USA), TGF-β1 (R&D Systems, Minneapolis, MN, USA), TGF-β inducible gene-h3 (βig-h3, Proteintech, Chicago, IL, USA), MnSOD (Abcam), Smad2/3 (Cell signaling Technology, Danvers, MA, USA), B-cell lymphoma-2 (Bcl-2, Santa Cruz Biotechnology), Bcl2-associated X (Bax, Delta Biolabs, Gilroy, CA, USA), and β-actin (Sigma) were detected with specific antibodies. Immunoblotting images were analyzed with an image analyzer (Quantity One, Bio-Rad Technical Service Department, Hercules, CA, USA). Optical densities were obtained using the VH group as 100% reference and normalized with β-actin.
6
Cytosolic Protein Analysis Protocol
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For analysis of cytosolic proteins, cells were lysed in cell lysis buffer [50 mm HEPES (pH 7.5); 1 mm DTT, 150 mM NaCl, 1 mM EDTA, 0.1 % Tween 20, 10 % glycerol, 10 mm β-glycerophosphate, 1 mM NaF, 0.1 mm orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 0.1 mM PMSF] and were incubated at 4 °C for 30 min. Protein concentration was measured using Bio-Rad protein assay dye concentrate. Lysates (30 μg) were resolved electrophoretically on 10 % SDS-polyacrylamide gel and electrotransferred to a polyvinylidine difluoride membrane (Bio-Rad) using a tank blot procedure (Bio-Rad Mini Protean II). The membranes were incubated with the following primary antibodies: Heme oxygenase-1 (HO-1) (Santa Cruz, Cat # sc-10789), cleaved caspase-3 (Cell Signaling, Cat # 9661), Bax (Santa Cruz Biotechnologies, Cat # 20067), Bcl2 (BD-Transduction Laboratories, Cat # 551052), β-actin (Cell Signaling Technologies, Cat # 4967), NOX4 (Abcam, Cat # ab60940), Mn-SOD (Abcam, Cat # 13533), Cu/Zn SOD (Abcam, Cat #13498), COX IV (Abcam, ab14744) and 1:1000 dilutions of respective horseradish peroxidase-linked F(ab) fragment secondary antibody (Amersham Corp., Piscataway, NJ) for 1 h. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection system (Amersham) as described previously [20 (link), 22 (link)].
7
Oxidative Stress Protein Profiling in HUVEC
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HUVEC were scraped on ice in a lysis buffer and centrifuged for 15 minutes at 13,000 rpm to recover the proteins. The lysis buffer used for protein extraction is made by mixing different compounds: 20mM Hepes, pH 7.4, 1% Triton X-100, 100mM NaCl, 50mM NaF, 10mM β-glycerophosphate, 1mM phenylmethylsulfonyl fluoride (PMSF), 1mM sodium orthovanadate, protease inhibitor cocktail (Roche Diagnostics, Basilea, Switzerland). Primary specific antibodies: PRX-SO3 (Abcam, ab16830), PRX6 (Abcam, ab59543), GPX1 (Abcam, ab22604), Catalase (Sigma-Aldrich, c0979), MnSOD (Abcam, ab13533), Cu/ZnSOD (Abcam, ab13498) and eNOS (Santa Cruz Biotechnology, sc-653), COX-1 (Santa Cruz Biotechnology, sc-19998), COX-2 (Santa Cruz Biotechnology, sc-19999), PGIS (Santa Cruz Biotechnology, sc-20933), TXAS (Santa Cruz Biotechnology, sc-79181), Caspase-3 (Cell Signaling, 9662), NLRP3 (Novus, NBP2-12446).2 Finally, β-actin (Sigma-Aldrich, a1978) (Sigma-Aldrich) as loading control.2 Detection was performed using peroxidase-linked secondary antibodies (anti-mouse (Sigma-Aldrich, NA931V), anti-goat (Santa Cruz Biotechnology sc2020), anti-rabbit (Sigma-Aldrich, NA934V).
Protein carbonyls were measured using the OxyBlot™ protein oxidation kit (Merck, Massachusetts, USA) following the manufacturer’s specifications.
Protein carbonyls were measured using the OxyBlot™ protein oxidation kit (Merck, Massachusetts, USA) following the manufacturer’s specifications.
8
Molecular Mechanisms of Neuroprotection
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GP17 (molecular weight = 947.154; purity > 98%) was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). The positive control drug NBP was obtained from CSPC NBP Pharmaceutical Co., Ltd. Triphenyltetrazolium chloride (TTC) was purchased from Sigma–Aldrich (MO, United States). Primary antibodies against p62/SQSTM1, LC3-B, BNIP3, NAMPT, SIRT1, SIRT2, MnSOD, PGC-1α, FOXO3 and p-FOXO3 were obtained from Abcam (Cambridge, UK). Primary antibodies against Hif1α and Beclin1 were obtained from Proteintech (Wuhan, China). A primary antibody against SIRT3 was obtained from Cell Signaling Technology (MA, USA). The inducer RAP and the inhibitors 3-MA and 2-ME were obtained from MedChemExpress (New Jersey, USA). The inhibitor AGK-7 was obtained from Abcam (Cambridge, UK). ELISA kits for IL-6, TNF–α, MCP-1, T-AOC and 4-HNE were acquired from HaiTai TongDa Sci Tech, Ltd. (Beijing, China).
9
Protein Expression Analysis in Kidney Tissues
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The protein concentrations of frozen kidney tissues were measured using a BCA protein assay (Pierce Biotechnology, Rockford, IL, USA). For immunodetection, the blot was incubated overnight with the primary antibodies against the following proteins: MnSOD (diluted 1:2,000, Abcam, Cambridge); catalase (1:2,000, Abcam); Bax (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); Bcl-2 (1:100, Santa Cruz Biotechnology); transforming growth factor (TGF)-β1 (1:4,000, R&D Systems, MN, USA); PI3K (1:1,000, Abcam); Akt (1:1,000, Cell Signaling Technology, Danvers, MA, USA), phospho-Ser473 Akt (1:1,000, Cell Signaling Technology); FoxO3a (1:1,000, Cell Signaling Technology); and phospho-Ser253 FoxO3a (1:1,000, Cell Signaling Technology). The protein bands were visualized using a chemiluminescence detection system (ImageQuant LAS 4000 mini, GE Healthcare, Piscataway, NJ, USA). Band intensities were determined with Image-Pro Plus software (Media Cybernetics Inc., Rockville, MD, USA).
10
D-Galactose-Induced Oxidative Stress Model
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D-gal was purchased from Sigma-Aldrich (St. Louis, USA). AOS was obtained from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China). The commercial kits used to measure blood urea nitrogen (BUN), serum creatinine (Scr), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were supplied by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The kit used to extract cytoplasmic and nuclear proteins was provided by Kangwei Century Bioengineering Institute (Beijing, China). The Nrf2, HO-1, NQO1, and GAPDH antibodies were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The Lamin B rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The p53, p21, Cu/Zn-SOD, Mn-SOD, and CAT antibodies were supplied by Abcam Inc. (Cambridge, UK). All the other chemicals and reagents were purchased from standard commercial suppliers if not otherwise stated.
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