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Figure Lengend Snippet: MCT induced 4-HNE accumulation in rat lung tissues. (A) 4-HNE was detected in pulmonary arterial smooth muscle cells (PASMCs) in the lungs of rats with MCT-induced PAH. Representative immunofluorescence images of lung sections stained with anti-4-HNE antibodies (green) and anti-α-SMA antibodies (red), at 0, 2, and 4 weeks post-MCT stimulation. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Compared with 0 week rats, 4-HNE levels in the pulmonary arterial media increased in rats with MCT-induced PAH, in terms of the percent nuclear localization. (B) 4-HNE expression in the lung tissue lysates, as determined by western blotting. (C) Quantification of protein levels from (B), relative to the control. * P <0.05 (n=3). (D) 4-HNE levels in tissue lysates measured by ELISA. Data are presented as the mean±SD (n=6 in each group). * P <0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used for western blotting: monoclonal antibodies against rabbit anti-cyclin D1, anti-c-Myc, anti-matrix metalloprotein (MMP)-9 and anti-MMP-2 (1:1000 dilution; Cell Signaling Technology, USA); polyclonal antibodies against rabbit anti-nuclear factor-kappa B (NF-κB) p65, anti-phospho-NF-κB p65, anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), and anti-phospho-IκBα (1:1000 dilution; Cell Signaling Technology, USA); monoclonal antibodies against mouse anti-4-HNE monoclonal antibody and mouse anti-ALDH2 (1: 500 dilution; Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Diabetes Mellitus Aggravates Hemorrhagic Transformation after Ischemic Stroke via Mitochondrial Defects Leading to Endothelial Apoptosis

doi: 10.1371/journal.pone.0103818

Figure Lengend Snippet: A: Cell proliferation (n = 7). B: ATP contents (n = 10). C: Mitochondrial membrane potential, determined by tetramethyl rhodamine methyl ester (TMRM) intensity (n = 10). The scale bar indicates 100 µm. D: reactive oxygen species (ROS) levels (n = 10). E: Binding of 4-hydroxy-2-nonenal (4-HNE), an indicator of lipoperoxidation by ROS. F: Enzymatic activities of MMP-2 and MMP-9 by gelatin zymography (n = 3). All data are expressed as mean ± SEM (shown as ratio to 5.5 mM). *P<0.05, **P<0.01 vs. 5.5 mM (Dunnet's test). HBMVECs, human brain microvascular endothelial cells.

Article Snippet: For immunoblotting, the following primary antibodies were used: mouse anti-Active Caspase-3 antibody (1∶1000 dilution; Genlantis, San Diego, CA, USA), mouse anti-Caspase-7 antibody (1∶1000 dilution; MBL, Nagoya, Japan), mouse anti-AIF antibody (1∶1000 dilution; Santa Cruz, CA, USA), mouse anti-cytochrome c antibody (1∶200 dilution; Santa Cruz), mouse anti-β3-Tubulin antibody (1∶1000 dilution; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-c-Jun antibody (1∶1000 dilution; Cell Signaling Technology), mouse anti-4-hydroxy-2-nonenal antibody (1∶10 dilution: JaICA, Shizuoka, Japan), mouse anti-HSP60 antibody (1∶1000 dilution; StressMarq Biosciences, Victoria, BC, Canada), rabbit anti-VDAC1 antibody (1∶100 dilution; Abgent, Inc., San Diego, CA, USA), and β-actin mouse monoclonal antibody (1∶5000 dilution; Sigma-Aldrich).

Techniques: Binding Assay, Zymography

Journal: PLoS ONE

Article Title: Renal Impairment with Sublethal Tubular Cell Injury in a Chronic Liver Disease Mouse Model

doi: 10.1371/journal.pone.0146871

Figure Lengend Snippet: ( A ) Representative images of 4-hydroxy-2-nonenal (4-HNE) immunostaining in the kidneys of control and DDC-fed mice at days 1, 3, and 7. 4-HNE staining on kidney tissue was digitally analyzed and presented as percentage of positive staining area per field; control ( white bar ) and DDC-fed mice ( black bar ) at days 1, 3, and 7. Scale bar = 50 μm. ( B ) Representative images of 3-nitrotyrosine (3-NT) immunostaining in the kidneys of control and DDC-fed mice at days 1, 3, and 7. The ratio of 3-NT-positive cells to total tubular cells are presented as a percentage; control ( white bar ) and DDC-fed mice ( black bar ) at days 1, 3, and 7. Scale bar = 50 μm. Data are presented as means ± SEM. * P < 0.05 compared with control mice.

Article Snippet: After rinsing and the blocking process by 1% bovine serum albumin and 0.1% cold water fish-skin gelatin for 30 min at room temperature, slides were treated with negative control reagents or the following optimally diluted primary antibodies: anti-Ly-6G/-6C (rat monoclonal, 1:50; Abcam, Cambridge, MA, USA), anti-leukocyte common antigen (LCA) (mouse monoclonal, 1:200; Dako, Glostrup, Denmark), anti-Ki67 (mouse monoclonal, 1:100; Novocastra, New Castle, UK), anti-4-hydroxy-2-nonenal (4-HNE) (rabbit polyclonal, 1:500; Merck Millipore, Darmstadt, Germany), and anti-3-nitrotyrosine (3-NT) (rabbit polyclonal, 1:100; Merck Millipore) for 1 hour at room temperature.

Techniques: Immunostaining, Staining