Immunohistochemical studies were performed in sections of paraffin-embedded joints from lean and db/db mice (mice with mutated receptor of leptin) obtained from a previous study of our group [36 (link)]. Samples were deparaffinised, cleared with xylene, and hydrated in a series of increasing grade alcohol. Heat-mediated antigen retrieval was performed in citrate buffer (pH 6.0; S2369; Dako, Agilent Technologies Spain S.L., Barcelona, Spain) for Nrf-2 detection or in ethylenediaminetetra-acetic acid (EDTA) buffer (pH 9.0; S2367; Dako) for CBS and CSE detection. Thereafter, peroxidase blocking solution (Dako) was used to block endogenous peroxidase activity. Then, slides were washed with phosphate buffer solution and incubated with primary antibodies against Nrf-2 (1:200; sc-13032; Santa Cruz Biotechnology), CBS (1:400; ab54883; Abcam), and CSE (1:400; ab54573; Abcam). Antigen–antibody interactions were determined with the rabbit/mouse peroxidase/DAB DAKO REAL EnVision Detection Kit (K5007; Dako). Sections were counterstained with haematoxylin and eosin. Slides were dehydrated in graded alcohol, cleared in xylene, and mounted in DePeX (Dako). Slides were visualised in an Olympus Dx61 optical microscope (Olympus España S.A.U., Barcelona, Spain). Staining intensity was quantified using ImageJ software.
Sc 13032
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-13032 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for use in scientific research and experimentation. The core function of this product is to provide a standardized and controlled environment for various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (2 mentions)
Sc 365962, by Santa Cruz Biotechnology (2 mentions)
Ab54883, by Abcam (1 mentions)
S2369, by Agilent Technologies (1 mentions)
S2367, by Agilent Technologies (1 mentions)
Dako real envision detection kit, by Agilent Technologies (1 mentions)
Dx61 optical microscope, by Olympus (1 mentions)
Peroxidase blocking solution, by Agilent Technologies (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Crystal mount, by Electron Microscopy Sciences (1 mentions)
Image pro plus v6, by Media Cybernetics (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Leica dmi8 confocal microscope, by Leica camera (1 mentions)
Sulfanilamide, by Merck Group (1 mentions)
Sc 1747, by Santa Cruz Biotechnology (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Sc 109, by Santa Cruz Biotechnology (1 mentions)
Sc 7884, by Santa Cruz Biotechnology (1 mentions)
Modified eagle s medium mem, by Thermo Fisher Scientific (1 mentions)
17 protocols using sc 13032
1
Immunohistochemical Analysis of Oxidative Stress Markers in Obese Mice
2
Immunoblot and Immunofluorescence Analysis
3
Quantification of NRF2 Expression in HCC
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Human HCC and AT samples were fixed in formalin (10%, pH 7.4) and embedded in paraffin wax. Serial sections of 4 μm thickness were cut and mounted on charged glass slides, and then stained with haematoxylin and eosin (H&E) or used for IHC staining as described previously (Pi et al, 2009 (link)). Rabbit polyclonal antibody against NRF2 (sc-13032, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at dilutions of 1 : 500. After counterstaining slides with H&E or IHC, cover slips were placed over the slides using Crystal Mount (Electron Microscopy Sciences, Hatfield, PA, USA). Quantitative analysis of NRF2 staining was carried out using the Image-Pro Plus v6.0 image analysis software (Media Cybernetics, Bethesda, MD, USA). Integrated optical density (IOD) value of NRF2 immunostaining was measured in five randomly acquired areas of one visual field in HCC and AT. Mean of IOD was used for further analyses.
4
Nrf2 Nuclear Translocation Assay
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For examining Nrf2 nuclear translocation, CD3+T cells were isolated from spleen of C57BL/6 mice and activated by plate bound anti-CD3 (5 µg/ml) and anti-CD28 (1 µg/ml) in the presence of DMF or DMSO for 3 h. The cells were harvested and fixed in 4% paraformaldehyde for 15 min, then permeabilized with 0.2% Triton X100 for 10 min, and blocked with 2% BSA for 30 min. Sample were incubated overnight with an anti-Nrf2 antibody (sc-13032, Santa Cruz, CA, USA) in 0.5% BSA. After three washes with PBS, cells were stained with Alexa Fluor 488 goat antirabbit IgG (Molecular Probes, USA). Cell nuclei were stained with DAPI. The fluorescent images were captured with the Leica DMi8 confocal microscope (Leica, Wetzlar, Germany).
5
Immunomodulatory Molecule Detection
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Fetal bovine serum (FBS) and modified Eagle’s medium (MEM) were purchased from Gibco (Grand Island, N.Y, USA), and phosphate-buffered saline (PBS), Western Breeze Chromogenic Kit anti-mouse, anti-rabbit, and anti-goat, and LysoTracker Green DND-26 were bought from Invitrogen (Carlsbad, CA, USA). Kit lactic dehydrogenase-based, sulfanilamide, H3PO4, and N-1-naphthylethylenediamine dihydrochloride were obtained from Sigma-Aldrich, St. Louis, MO, USA. Antibodies for IL-1β (H153): sc-7884, IL-2 (C2-1-hIL-2): sc-32295, IL-6 (E-4): sc-28343, IL-8 (807): sc-52870, IL-18 (H-173): sc-7954, tumor necrosis factor-alpha (TNF-α) (N-19): sc-1350, cyclooxygenase-2 (COX-2) (M-19): sc-1747, nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) (H-300): sc-13032, matrix metalloproteinase-2 (MMP-2) (H-76): sc-10736, matrix metalloproteinase-9 (MMP-9) (M-17): sc-6841, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 (A): sc-109 and β-actin (C4): sc-47778 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Invitrogen (Carlsbad, CA, USA). All of the other chemicals used were of analytical grade and were purchased from Sigma (St. Louis, MO, USA).
6
Western Blot Analysis of Hrd1 and Nrf2
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Cells were washed twice in ice-cold PBS, and then solubilized in RIPA lysis buffer (Vazyme). Proteins extracted were quantified using a Bicinchoninic Acid Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology). Equal amounts of each protein sample (30 µg/lane) were subjected to 10% SDS-PAGE and were transferred to polyvinylidene fluoride membranes, which were blocked with 5% skimmed milk at room temperature for 1 h. Members were incubated overnight at 4°C with antibodies targeting Hrd1 (cat. no. ab170901; 1:2,000; Abcam), Nrf2 (cat. no. sc-13032; 1:2,000; Santa Cruz Biotechnology, Inc.) and β-actin (cat. no. 58169, 1:1,000; Cell Signaling Technology, Inc.), followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab6721; 1:3,000; Abcam) for 1.5 h at room temperature. β-actin was used as a loading control. Protein bands were visualized by enhanced chemiluminescence (cat. no. WBKLS0500; EMD Millipore). ImageJ 1.45 software (National Institutes of Health) was used to perform densitometric analysis of each band.
7
Immunofluorescence Assay for NRF2 and EMT Markers
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RT4-WT and Crispr/Cas9 NRF2-KO cells were washed with warmed PBS twice, followed by fixation with chilled 4% paraformaldehyde (Sigma) in PBS for 15 min, all reagents were kept cold past this point and incubation was performed at room temperature unless stated otherwise. Permeabilization was performed with 1% Triton X-100 in PBS for 10 minutes followed by a blocking step with 3% bovine serum albumin (BSA) in PBS for 30 minutes. The cells were incubated overnight at 4°C with the primary antibodies and then incubated in the dark for 2 hours against the secondary antibodies. Primary antibodies used were NRF2 (1:50; cat. # sc-13 032, Santa Cruz Biotechnology), HO-1 (1:100; cat. # ab13248, Abcam), E-cadherin (1:50; cat. # M3612, Dako), and ZEB1 (1:50; cat # ab124512, Abcam). Secondary antibodies used were Alexa conjugated secondary antibodies (1:250, Life technologies). The antibodies were all diluted in 3% BSA solution. Wells were washed 3 times with PBS in between each step. Cells were examined using a laser scanning confocal microscope (Leica TCS SP8; Leica Microsystems, Wetzlar, Germany).
8
Immunohistochemical Analysis of NRF2
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PC12 cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed three times with PBS. The cells were permeabilized in 0.1% TritonX-100 (Sigma—Aldrich St. Louis, MO, USA) for 15 min and blocked in 1% BSA for 1 h at room temperature. After being washed, the cells were incubated with a 1:200 dilution of primary NRF2 antibody (sc-13032, Santa Cruz Biotechnology, USA, 1:200) at 4 °C overnight and incubated with appropriate secondary antibody (sc-13032, goat anti-rabbit IgG-PE, Santa Cruz Biotechnology, USA, 1:300) for 1 h at room temperature. Cell nuclei were stained with DAPI. The images were acquired with the fluorescence microscope (Nikon, Japan).
9
Synthesis and Characterization of RRx-001
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RRx-001 was obtained from ATK Aerospace Systems [10 ]. The synthesis and characterization of RRx-001 is reported in detail elsewhere [8 –12 (link)]. For in vitro cell culture experiments, RRx-001 was dissolved in DMSO and then diluted with growth medium with a final concentration of DMSO at < 0.05%. For animal experiments, RRx-001 formulation was prepared by dissolving 10 mg RRx-001 in 0.5 mL DMA-PEG 400 (1:2) and then diluting with double distilled water to obtain a 2 mg/mL solution for injection.
The pcDNA-ARE-FLUC and pcDNA-CMV-RLUC-mRFP vectors were constructed in our lab. Nrf2-specific small interfering RNA (Nrf2-siRNA, sc-37049), scrambled siRNA (sc-37007), transfection reagents (sc-29528 and sc-36868), and antibodies against Nrf2 (sc-13032), HO-1 (sc-10789), NQO1 (sc-393736), Δ-actin (sc-130656) and lamin-B (sc-365962) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA.
The pcDNA-ARE-FLUC and pcDNA-CMV-RLUC-mRFP vectors were constructed in our lab. Nrf2-specific small interfering RNA (Nrf2-siRNA, sc-37049), scrambled siRNA (sc-37007), transfection reagents (sc-29528 and sc-36868), and antibodies against Nrf2 (sc-13032), HO-1 (sc-10789), NQO1 (sc-393736), Δ-actin (sc-130656) and lamin-B (sc-365962) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA.
10
Reagents for Nrf2 and NF-κB Signaling
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TLR4-specific E. coli LPS was purchased from Alexis Biochemicals (San Diego, CA, USA). Mouse INF-γ was supplied by R&D Systems (Minneapolis, MN, USA). All antibodies used in this study, including antibodies to Nrf2 (SC-13032), the p65 subunit of nuclear factor-κB (NF-κB; SC-8008), lamin B (SC-365962) and hnRNP (SC-10030R) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All chemicals and reagents, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sulforaphane (SFN) and N-acetyl cysteine (NAC) were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless indicated otherwise.
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