Paraffin-embedded liver sections (5 µm) were stained for 4-hydroxynonenal (4-HNE) protein adducts and iNOS using a polyclonal antibody (4-HNE: AG Scientific; iNOS: Affinity BioReagents), as described previously (17) . Paraffin-embedded duodenal sections (4 µm) were stained for occludin (Invitrogen Corporation), zonula occludens 1 (ZO-1; Life Technologies GmbH) and hydroxyindole-O-methyltransferase (HIOMT; Biozol Diagnostica Vertrieb GmbH) using polyclonal primary antibodies, as described previously (17) . In brief, specific binding of primary antibody to the target protein was detected by incubating sections with a peroxidase-linked secondary antibody and diaminobenzidine (Peroxidase Envision Kit; Dako). Photos of eight fields of each tissue section (200× of each liver tissue section; 400× of occludin staining and 200× of HIOMT staining of duodenum tissue) were captured and the extent of staining in the sections was defined as percentage of microscopic field within the default colour range determined by the analysis system incorporated in the microscope (Leica DM4000 B LED). Mean values of eight sections were used as the score of the sample.
4 hne
Manufactured by AG Scientific
Sourced in United States
4-HNE is a highly reactive aldehyde that is commonly used as a biomarker for oxidative stress. It is a product of lipid peroxidation and can be detected in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase envision kit, by Agilent Technologies (4 mentions)
Dm4000 b led, by Leica (3 mentions)
4 hne, by Santa Cruz Biotechnology (2 mentions)
Myd88, by Santa Cruz Biotechnology (2 mentions)
F4 80, by Abcam (2 mentions)
Occludin and zo 1, by Thermo Fisher Scientific (2 mentions)
Occludin, by Thermo Fisher Scientific (1 mentions)
4 hne, by Abcam (1 mentions)
Applications suite, by Leica (1 mentions)
6 protocols using 4 hne
1
Immunohistochemical Analysis of Liver and Duodenum
2
Hepatic and Intestinal Immunohistochemistry
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Paraffin-embedded liver tissue sections (4 µm) were stained for myeloid differentiation primary response gene 88 (MyD88), 4-hydroxynonenal (4-HNE) protein adducts, inducible nitric oxide synthase (iNOS), F4/80 and cluster of differentiation 8α (CD8α)-positive cells using polyclonal antibodies (MyD88: Santa Cruz Biotechnology; 4-HNE: AG Scientific; iNOS: Affinity BioReagents; F4/80: Abcam; CD8α: Santa Cruz Biotechnology) as previously detailed (15, 16) . Paraffin-embedded duodenal tissue sections (4 µm) were stained for the tight junction proteins occludin and zonula occludens 1 (ZO-1) using polyclonal primary antibodies (Life Technologies GmbH) as described previously (11, 17) . In brief, to detect the binding of target protein to the specific primary antibody, tissue sections were incubated with a peroxidase-linked secondary antibody and diaminobenzidine (Peroxidase Envision Kit; Dako). Pictures of eight fields of each tissue section (×200 of each liver tissue; ×400 of each duodenum tissue) were captured using a digital camera integrated in a microscope (Leica DM4000 B LED), and the extent of staining in tissue sections was defined as per cent of field area within the default colour range determined by an analysis system incorporated in microscope. Mean values of eight sections were used per tissue section to determine means.
3
Comprehensive Histological Assessment of NAFLD
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Liver histology was assessed using the NAFLD activity score (NAS), as described previously [24 (link)]. Staining and counting of the number of neutrophilic granulocytes and assessment of hepatic fibrosis were carried out as described previously [25 (link)]. Liver sections were stained for F4/80, iNOS and 4-hydroxynonenal protein adducts (4-HNE) using polyclonal antibodies (F4/80: Abcam, Cambridge, UK; iNOS: Affinity BioReagents, Rockford, USA; 4-HNE: AG Scientific, San Diego, USA) and staining was evaluated as described before [6 (link)]. Paraffin-embedded sections of proximal small intestine (4 µm) were stained and analysed for the tight junction proteins occludin, zonula occludens 1 (ZO-1) and hydroxyindole-O-methyltransferase (HIOMT), respectively, using polyclonal primary antibodies (occludin and ZO-1: Invitrogen, CA, USA; HIOMT: Biozol Diagnostica GmbH, Germany) as previously described [19 (link)].
4
Immunohistochemical Analysis of Liver and Intestinal Markers
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Immunohistochemical staining methods were used to measure concentration of 4-HNE, iNOS and MyD88 in paraffin embedded liver sections as well as of MMP13, Occludin and ZO-1 in small intestinal tissue sections as previously described7 (link),19 (link),22 (link). In brief, after treating sections with citrate buffer (MMP13) or incubating with protease (occludin, ZO-1) and blocking tissue sections with bovine serum albumin solution (iNOS, MMP13, MyD88), they were incubated with specific primary antibodies (4-HNE: AG Scientific, San Diego, CA, USA; iNOS: Thermo Fisher Scientific, Waltham, MA, USA; MMP13: LifeSpan BioSciences, Seattle, WA, USA; MyD88: Santa Cruz Biotechnology, Dallas, TX, USA; Occludin and ZO-1: Invitrogen, Carlsbad, CA, USA). Subsequently, sections were incubated with peroxidase-linked secondary antibodies followed by diaminobenzidine (Peroxidase Envision Kit, DAKO, Hamburg, Germany). Staining was evaluated using a camera integrated in a microscope (Leica DM4000 B LED, Leica, Wetzlar, Germany) and an analysis system (Leica Applications Suite, Leica, Wetzlar, Germany) as described previously19 (link),42 (link).
5
Immunohistochemical Analysis of Liver Oxidative Stress
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Paraffin-embedded liver samples were cut into 4 µm sections and stained to detect inducible nitric oxide synthase (iNOS), 4-hydroxynonenal protein adducts (4-HNE) and 3-nitrotyrosine protein adducts (3-NT) using monoclonal (3-NT: Santa Cruz, USA) and polyclonal (4-HNE: AG Scientific, USA, iNOS: Affinity BioReagents, USA) antibodies. To detect specific binding of primary antibodies, tissue sections were incubated with a peroxidase linked secondary antibody and diaminobenzidine (Peroxidase Envision Kit; Dako, Hamburg, Germany). Using an image acquisition and analysis system incorporated in the microscope, the extent of staining in liver sections was defined as percent of the field area within the default color range determined by the software. To determine means, data from eight fields of each tissue section (200× magnification) were used.
6
Immunohistochemical Analysis of Liver and Intestine
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4-Hydroxynonenal (4-HNE) protein adducts in paraffin embedded liver sections (4 µm) and tight junction proteins occludin and zonula occludens (ZO)-1 in intestinal tissue (4 µm sections) were stained using polyclonal antibodies (4-HNE: AG Scientific, USA; occludin: Invitrogen, USA; ZO-1: Invitrogen, USA). Extent of staining was determined as described previously (Sellmann et al. 2015 (link)). Briefly, data from eight randomly selected microscopic fields (200× for liver and 400× for intestine) of each tissue section were used to determine staining intensity.
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