Whole brains from each rat in each treatment group were removed and embedded in Optimal Cutting Temperature (OCT) compound and frozen in liquid nitrogen. Frozen tissue blocks were sectioned at 10 μm, mounted on positive-charged slides, and air-dried for 10 min. Sections were fixed in cold acetone (−20 °C) for 10 min, dried for 20 min, and incubated with streptavidin–horseradish peroxidase (HRP) polymer kit (Biocare Medical) for 10 min, stained with DAB chromogen (Vector Labs), and then counter-stained with hematoxylin (Vector Labs). The streptavidin binds to the biotin on the anti-DMPO probe if present. For immunohistochemistry (IHC) levels of inducible nitric oxide synthase (iNOS), a rabbit polyclonal anti-rat antibody against iNOS (ab15326; Abcam, Cambridge, MA) was used. For IHC levels of nuclear factor erythroid 2-related factor 2 (Nrf2), a rabbit polyclonal anti-rat antibody against Nrf2 (ab31163; Abcam, Cambridge, MA) was used. For IHC levels of 3-nitrotyrosine (3-NT), a mouse monoclonal antibody against 3-NT (ab61392; Abcam, Cambridge, MA) was used. For IHC levels of malondialdehyde (MDA), a rabbit polyclonal antibody against MDA-PC (protein carrier) (ab6463; Abcam, Cambridge, MA) was used.
Ab6463
Manufactured by Abcam
Sourced in United Kingdom
Ab6463 is an antibody product manufactured by Abcam. It is a primary antibody designed for use in various laboratory applications. The core function of this product is to detect and bind to a specific target antigen. Further details on the intended use or performance characteristics of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab46545, by Abcam (2 mentions)
Dab chromogen, by Vector Laboratories (1 mentions)
Ab15326, by Abcam (1 mentions)
Ab61392, by Abcam (1 mentions)
Ab31163, by Abcam (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Fluorophore conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Nf κb, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Ab9391, by Abcam (1 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
Ab201340, by Abcam (1 mentions)
Ab53444, by Abcam (1 mentions)
Ab48506, by Abcam (1 mentions)
Sc 1747 r, by Santa Cruz Biotechnology (1 mentions)
Affinipure goat anti rabbit igg h l antibody, by Thermo Fisher Scientific (1 mentions)
13 protocols using ab6463
1
Immunohistochemical Analysis of Oxidative Stress Markers
2
Histological Analysis of Mouse Liver and Lung
3
Placental Protein Analysis by Western Blot
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Homogenized placental samples were lysed in RIPA buffer with protease and phosphatase inhibitors. Protein concentration was determined by Bradford assay (Bio-Rad, Hercules, USA). A quantity of 30–40 µg total protein per lane was separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, USA). Blocked membranes were incubated with primary antibodies such as β-actin (1:1500, Sigma), MDA (1:400, ab6463, Abcam), NFκB (1:500, ZS-109), AT1R (1:500, ab9391, Abcam), p38 (1:500, Cell Signaling Technology, Beverly, MA, USA), and c-Jun N-terminal kinase (JUK, 1:500, Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C. The specificity of the immune response was detected without adding the first antibodies. After hybridization with a secondary antibody (1: 2000), the target proteins were finally detected using ECL Western Blotting Detection Reagents (Thermo Scientific Pierce, Rockford, IL, USA).
4
Immunohistochemical Analysis of Kidney Samples
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Kidney samples were fixed in 10% neutral-buffered formalin and processed to 5 μm thick sections for immunohistochemistry. Tissue sections were deparaffinized, rehydrated in ethyl alcohol, and washed in tap water. Antigen retrieval was performed using 10 mM sodium citrate buffer for 20 min in a Black & Decker vegetable steamer. Slides were blocked with 10% donkey serum for 30 min at RT, and then washed with PBS. Primary antibodies against xCT (Novus Biologicals), GPX4 (Novus Biologicals), 4-hydroxynonenal (4-HNE; ab46545, Abcam), or MDA (ab6463, Abcam) were diluted to the appropriate concentration with 2% casein in bovine serum albumin, added to the slides, and incubated overnight at 4 °C. After washing, the secondary antibody (Dako, Carpinteria, CA, USA) was applied for 1 h at RT. Diaminobenzidine was added for 2 min, and sections were counterstained with hematoxylin. A semiquantitative score for staining intensity was obtained by examining at least five fields per section under ×20 magnification and with digital image analysis (MetaMorph version 4.6r5, Universal Imaging Corp., Downingtown, PA, USA).
5
Immunohistochemical Analysis of Oxidative Markers
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Immunohistochemical analysis was performed as described elsewhere [10 ]. Antibodies and dilutions used in this study include anti–4-hydroxynonenal (1:150 dilution; cat. # ab48506, Abcam), anti-malondialdehyde (1:300; cat. # ab6463, Abcam), anti-CD68 (1:50; cat. # ab201340; # ab53444, Abcam), and anti-COX2 (1:50; cat. # ab201340, Abcam and 1:50; # sc-1747R, Santa Cruz, USA), antibodies; with the respective secondary antibodies: a cyanine 3–conjugated goat anti-mouse IgG (H+L) cross-adsorbed antibody, (1:500; cat. #M30010 Invitrogen, USA) or a GFP-conjugated AffiniPure goat anti-rabbit IgG (H+L) antibody (1:1000; cat. #111-095-003, Jackson AB, USA) and mouse anti-rabbit IgG-CFL 555: sc-516249, Santa Cruz, USA). The slices were coverslipped with the Fluoro-shield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; cat. # F6057, Sigma-Aldrich, USA) and examined under an Axioplan 2 microscope (Zeiss, Germany).
6
Immunohistochemical Analysis of Oxidative Stress and Apoptosis
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The 5–6 μm thick cross-sections prepared from paraffin blocks were placed on polylysine-coated microscopic slides. Routine immunohistochemical staining was performed according to the method of Kuloğlu and Aydin.12 (link) Primary antibodies (rabbit polyclonal anti-malondialdehyde antibody, ab6463; Abcam, Cambridge, UK, and CASP3 rabbit polyclonal immunoglobulin G, ab2302; Abcam) immunoreactivity were studied with avidin–biotin–peroxidase methodology. Ki-67 (monoclonal mouse, anti-human, clone MIB-1 code 7240, Dako Denmark A/S, Glostrup, Denmark) antibody was performed with automatic painting device (SN:712299; Ventana Medical Systems, Inc., Tucson, AZ, USA). Light microscopy and photography were done using an Olympus BX50 light microscopy (Olympus Corporation, Tokyo, Japan). A histoscore was derived from the distribution (0.1: <25%, 0.4: 26%–50%, 0.6: 51%–75%, 0.9: 76%–100%) and intensity (0: no staining, +0.5: very little staining, +1: little staining, +2: medium, +3: very strong) of staining immunoreactivity (histoscore = distribution × intensity).13
7
Protein Extraction for Western Blot Analysis
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Protein extraction for WB analysis was performed as reported by De Vreese and colleagues (2019) [38 (link)], using frozen brain tissue from bottlenose dolphin, fin whale, Cuvier’s beaked whale, striped dolphin and sperm whale specimens. Overnight incubation at 4°C followed, using polyclonal rabbit Apaf-1 (Enzo, #ADI-905-179-100, 1:1000), anti-DGK-ζ (MyBiosourse, #MBS2026991, 1:500), anti-Bcl-2 (Abcam, #ab196495, 1:1000), anti-MDA (Abcam, #ab6463, 1:1000) and anti-iNOS (Abcam, #ab15323, 1:250); a monoclonal recombinant rabbit anti-Aβ (ThermoFisher Scientific, #700254, 1:500); and monoclonal mouse anti-NF200 (Sigma-Aldrich, #N0142), anti-GAD67 (Sigma-Aldrich, #MAB5406, 1:2000), anti-GAP43 (Sigma-Aldrich, #MAB347, 1:1000), anti-TNFα (Santa Cruz, #sc-52746, 1:200), anti-QKI-7 (Antibodies-online, #ABIN1304925, 1:1000) and anti-synaptophysin (Dako; #M7315, 1:1000). After several washes in TBS-T, the membrane was incubated with an anti-rabbit peroxidase-conjugated secondary antibody (ThermoFisher Scientific, #32260) for 1 hour at room temperature. In order to visualize immunoreactive bands, a chemiluminescent detection kit (SuperSignal West Pico Chemiluminescent Substrate, ThermoFisher Scientific) and the iBright machine (ThermoFisher Scientific) were employed.
8
Immunohistochemical Analysis of Arterial Lipids
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9
Lipid Peroxidation Analysis in Atherosclerosis
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Lipid peroxidation in plasma of Pcyox1+/+/Apoe−/− and Pcyox1−/−/Apoe−/− mice after 8 weeks on HFD was assayed according to the manufacturer’s instructions using the Thiobarbituric Acid Reactive Substances Assay Kit (Cayman Chemicals), which measures levels of MDA, a byproduct of lipid peroxidation. Quantification was performed by fluorometric measurement (ex 530 nm, em 550 nm).
To assess the extent of lipid peroxidation in aortic root plaques, FFPE tissue sections were stained IHC with a rabbit polyclonal to MDA (ab6463, Abcam; 1 µg/mL). Sections were subjected to HIER in Dako target retrieval citrate buffer pH 6.0, treated with 3% H2O2, and blocked for non-specific staining by using Rodent Block M reagent (Biocare Medical). Sections were then incubated overnight at 4 °C with the primary antibody, followed by detection with rabbit-on-rodent HRP polymer detection system (Biocare Medical) according to the manufacturer’s instructions. Negative controls were prepared by the omission of the primary antibody. Sections were then processed and digitally captured as described above. Positivity to MDA was quantified with Axiovision as described for the other IHC analyses.
To assess the extent of lipid peroxidation in aortic root plaques, FFPE tissue sections were stained IHC with a rabbit polyclonal to MDA (ab6463, Abcam; 1 µg/mL). Sections were subjected to HIER in Dako target retrieval citrate buffer pH 6.0, treated with 3% H2O2, and blocked for non-specific staining by using Rodent Block M reagent (Biocare Medical). Sections were then incubated overnight at 4 °C with the primary antibody, followed by detection with rabbit-on-rodent HRP polymer detection system (Biocare Medical) according to the manufacturer’s instructions. Negative controls were prepared by the omission of the primary antibody. Sections were then processed and digitally captured as described above. Positivity to MDA was quantified with Axiovision as described for the other IHC analyses.
10
Histological Analysis of AAA Samples
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Samples of arterial wall and intra-luminal thrombus obtained from AAA patients were embedded in paraffin, and 4 μm cross-sections were cut. Ceroids were detected by direct observation of tissue by fluorescence microscopy (ceroids autofluoresce at 550 nm, producing a red signal). Immunohistochemistry was performed with antibodies against the following proteins: PRDX6 (ab16947, abcam), the lipid peroxidation marker MDA (ab6463, abcam), the neutrophil marker CD15 (Dako), and alpha smooth muscle actin (Dako). Sections were then incubated with the appropriate biotinylated secondary antibody and ABComplex, followed by staining with 3,30-diaminobenzidine (DAB), hematoxylin counterstaining, and mounting in DPX medium.
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