Liver tissues were lysed using RIPA lysis buffer (150 mM NaCl, 1% triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris–HCl) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Protein samples were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (GVS, Bologna, Italy). After blocking with 5% BSA or skim milk for 30 min, PVDF membranes were incubated overnight with primary antibodies against B-cell lymphoma 2 (Bcl-2, 1:2000, Cell Signaling Technology Danvers, MA), cleaved caspase-3 (1:2000, Cell Signaling Technology, Danvers, MA), Ly6G (1:2000, eBioscience, San Diego, CA ), 4-hydroxynonenal (4-HNE, 1:2000, Abcam, Cambridge, UK), NFκB p65 (1:2000, Cell Signaling Technology, Danvers, MA), IκBa (1:2000, Cell Signaling Technology, Danvers, MA) and GAPDH (1:10,000, Bioworld Technology, St. Louis Park, MN). The membranes were then incubated with HRP-labelled goat anti-mouse IgG (1:3000, BETHYL-Laboratories, Montgomery, TX) or goat anti-rabbit IgG (1:3000, BETHYL-Laboratories, Montgomery, TX) secondary antibodies for 1 h at room temperature. Total protein expression levels were normalized to GAPDH (Supplementary Figures ). The intensity of each protein band was analyzed using the ImageJ v.1.53e software (NIH, Bethesda, MD, USA, https://imagej.nih.gov/ij ).
4 hydroxynonenal 4 hne
Manufactured by Abcam
Sourced in United States, United Kingdom
4-hydroxynonenal (4-HNE) is a reactive aldehyde that is a biomarker for oxidative stress. It is a product of lipid peroxidation and can be used to measure oxidative damage in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Gapdh, by Bioworld Technology (2 mentions)
Collagen 1, by Abcam (2 mentions)
α smooth muscle actin α sma, by Merck Group (2 mentions)
Kim 1, by Abcam (2 mentions)
Galectin 3, by Abcam (2 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by Fortis Life Sciences (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Ab152, by Merck Group (1 mentions)
Mab318, by Merck Group (1 mentions)
Gp91phox antibody, by BD (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Xylenol orange, by Merck Group (1 mentions)
Hesperetin, by Merck Group (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
23 protocols using 4 hydroxynonenal 4 hne
1
Western Blot Analysis of Liver Proteins
2
Histological Analysis of Kidney Injury
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After harvesting, kidneys were immediately fixed in 4% paraformaldehyde and then embedded in paraffin. The tissues were sectioned and stained with hematoxylin & eosin (H&E) stain, periodic acid Schiff (PAS) stain, and Masson’s trichrome stain. Images were captured using a confocal microscope (Nikon, Tokyo, Japan). Tubular injury in PAS-stained sections was assessed and scored at a ×200 magnification using 10 randomly selected fields for each kidney as follows: 0, 0%; 1, ≤10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; and 5, 76–100% [18 (link)]. For IHC staining, the sections were incubated with primary antibodies against kidney injury molecule-1 (Kim-1; Abcam, Cambridge, MA, USA), neutrophil gelatinase-associated lipocalin (NGAL; Santa Cruz Biotechnology, Santa Cruz, CA, USA), 4-hydroxynonenal (4-HNE; Abcam), galectin-3 (Abcam), α-smooth muscle actin (α-SMA; Sigma-Aldrich), or collagen I (Abcam) overnight at 4 °C and then probed with a secondary antibody for 30 min at room temperature. All the sections were counterstained with hematoxylin. The percentage of positive staining was assessed in 5 randomly selected fields (×400 magnification) per each kidney using i-Solution Lite V.9.1 image analysis software (IMTechnology, Vancouver, BC, Canada). Galectin 3 positive cells or CD4 positive cells were counted in 5 randomly selected fields (×400 magnification) per each kidney.
3
Histological Assessment of Kidney Injury
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Kidneys were rapidly removed from each mouse and then fixed in 4% phosphate-buffered paraformaldehyde. The tissues were embedded in paraffin and then thin sections were made from the paraffin blocks. The sections were incubated with hematoxylin and eosin (H&E) stain and periodic acid Schiff (PAS) stain. Images were captured using the NIKON A1+ confocal microscope (Nikon, Tokyo, Japan). The degree of tubular injury was scored as previously described [18 (link)]. For immunohistochemical staining, the kidney sections were probed with antibodies against kidney injury molecule-1 (Kim-1; Abcam, Cambridge, MA, USA), neutrophil gelatinase-associated lipocalin (NGAL; Santa Cruz Biotechnology), 4-hydroxynonenal (4-HNE; Abcam), or Galectin-3 (Abcam).
4
Oxidative Stress Immunohistochemistry Protocol
5
Oxidative Stress and Apoptosis Evaluation
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β-NADP+, acrolein, hesperetin, rhodamine 123 and xylenol
orange were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
7-Amino-4-chloromethylcoumarin (CMAC) was purchased from Invitrogen (Waltham,
MA, USA). Antibodies were purchased as follows: β-actin and cleaved caspase
3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); cytochrome C, phospho-p38
(p-p38), phospho-JNK (p-JNK), cleaved PARP, phospho-p53 (p-p53), and horseradish
peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, Beverly, MA,
USA); 4-hydroxynonenal (4-HNE) (Abcam, Cambridge, UK);
peroxiredoxin-SO3 (Prx-SO3) (Abfrontier, Seoul,
Korea); 8-OH-dG (Millipore, Billerica, MA, USA) antibodies.
orange were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
7-Amino-4-chloromethylcoumarin (CMAC) was purchased from Invitrogen (Waltham,
MA, USA). Antibodies were purchased as follows: β-actin and cleaved caspase
3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); cytochrome C, phospho-p38
(p-p38), phospho-JNK (p-JNK), cleaved PARP, phospho-p53 (p-p53), and horseradish
peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, Beverly, MA,
USA); 4-hydroxynonenal (4-HNE) (Abcam, Cambridge, UK);
peroxiredoxin-SO3 (Prx-SO3) (Abfrontier, Seoul,
Korea); 8-OH-dG (Millipore, Billerica, MA, USA) antibodies.
6
Protein Expression Analysis in Muscle Tissue
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After extraction, protein concentrations were tested using a BCA protein assay kit (Pierce Co, Rockford, IL, USA). Equal amounts of protein were run in 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane and then incubated with the primary antibodies at 4 °C overnight: β-myosin heavy chain (β-MHC; 1:1000, Abcam, Cambridge, MA, USA), α-smooth muscle actin (α-SMA; 1:1000, Abcam), 4-hydroxynonenal (4-HNE; 1:1000, Abcam), p53 acetylation (1:1000, Abcam), p53 (1:1000; Cell signaling, Danvers, MA, USA), glutathione peroxidase 4 (GPX-4) (1:1000; Novus Bio, Littleton, CO, USA) and TGF-β1 (1:500; Santa Cruz, CA, USA). After incubation with secondary antibody (1:5000; Santa Cruz), the signals were visualized and analyzed through image acquisition and analysis software (Bio-Rad, Hercules, CA, USA).
7
Immunohistochemical Analysis of Neurodegeneration
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After behavioral tests, 5 out of 10 animals were intracardially perfused with 4% paraformaldehyde in PBS under the deep anesthesia (Na pentobarbital, 100 mg/kg). After perfusion, the brains were removed from the skull and post-fixed for 24 hr in the same fixative at 4°C, and were then cryoprotected in 30% sucrose prepared in phosphate buffer. Serial coronal sections of brain (30 μm) were cut with a freezing slide microtome (Thermo Fisher Scientific, Waltham, MA, USA). The sections were incubated overnight at 4°C with antibody to Aβ1–42 (1:2000, Covance, Berkeley, CA, USA) or 4-hydroxynonenal (4-HNE) (1:2000, Abcam, Cambridge, MA, USA). After multiple washings in PBS, the sections were incubated in biotinylated IgG (1:1000, Vector Laboratories Inc., Burlingame, California, USA) for 1 hr at room temperature. The sections were subsequently washed and incubated with avidin-conjugated peroxidase complex (ABC kit, Vector Laboratories Inc.) for 60 min. The immunocomplex was visualized by using 3,3’-diaminobenzidine tetrahydrochloride (Sigma-Aldrich Korea, Seoul, Korea) as the chromogen. Finally, the brain sections were rinsed and mounted on poly-glycine-coated slides and analyzed under the light microscopy.
8
Quantifying Mitochondrial Protein Expression
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Cells were homogenized in cell lysis buffer (30 mmol/L HEPES [pH 7.4], 1% NP-40, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 10% glycerol, and protease inhibitor cocktail [Sigma, St. Louis, MO]). The protein concentration was measured using a Micro BCA Protein Assay Reagent (Pierce, Rockford, IL). The protein extracts were resolved by SDS-PAGE and electrotransferred onto an Immobilon polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membranes were probed with the following primary antibodies: MnSOD; Tom20 (Santa Cruz Biotechnology); succinate dehydrogenase subunit A (SDHA), phospho–dynamin-related protein (DRP1) (Ser637), DRP1, mitofusin 1 (MFN-1), TXNIP, GLUT1, GAPDH, and tubulin (Cell Signaling Technology, Danvers, MA); 4-hydroxynonenal (4-HNE) (Abcam, Cambridge, MA); and glutathione peroxidase 4 (GPX4) (R&D Systems). Alexa Fluor anti-rabbit 680 (Invitrogen, Carlsbad, CA) and anti-mouse 800 (VWR International, West Chester, PA) were used as the secondary antibodies. Fluorescence was quantified by using the Odyssey imager (LI-COR Biosciences, Lincoln, NE).
9
Quantifying 4HNE and Ubiquitinated Proteins
10
Western Blot Analysis of Kidney Tissue
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Kidney tissues were lysed and homogenized using the radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl, pH 8.0, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 M NaF plus protease inhibitor cocktail [Sigma-Aldrich] and phosphatase inhibitor cocktail [Sigma-Aldrich]). Extracted protein samples were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (GVS S.p.A.). After blocking with 5% bovine serum albumin or skim milk for 30 minutes, the membranes were incubated with antibodies against α-smooth muscle actin (α-SMA, 1:20,000; Sigma-Aldrich), phosphorylated SMAD3 (p-SMAD3, 1:2,000; Abcam), p-SMAD2 (1:2,000; Abcam), t-SMAD 2/3 (1:2,000; Abcam), 4-hydroxynonenal (4-HNE, 1:2,000; Abcam), Ly6G (1:2,000; Fisher Scientific), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:10,000; Bioworld Technology) overnight at 4 °C. The membranes were then treated for 1 hour at room temperature with horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin G (IgG, 1:3,000; Bethyl-Laboratories) or HRP-labeled goat anti-rabbit IgG (1:3,000; Bethyl-Laboratories). Total protein expression levels were normalized to GAPDH. Band intensities were analyzed using ImageJ software.
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