Biochemical and histological analyses were performed as reported previously[19 (link)]. Plasma analytes included alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG) and total cholesterol (TC). Liver homogenates were analyzed for TG and TC. Paraformaldehyde-fixed liver pre- and post-biopsies were paraffin-embedded, sectioned, and stained with hematoxylin-eosin (Dako, Glostrup, Denmark), Picro-Sirius red (Sigma-Aldrich, Broendby, Denmark), anti-type I collagen (Col1a1; Southern Biotech, Birmingham, AL), or anti-galectin-3 (Biolegend, San Diego, CA, United States). The NAFLD activity score (NAS) and fibrosis staging system was applied to liver pre-biopies and terminal samples (drug treatment experiments) or only terminal samples (disease progression experiment) for scoring of steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis outlined by Kleiner et al[31 (link)]. All histological assessments were performed by a pathologist blind to treatment. Because all treatment paradigms affected total liver weight, quantitative data on liver biochemistry (liver TG, TC) and histology (liver lipid, galectin-3, Col1a1) were expressed as whole-liver amounts by multiplying individual terminal liver weight with the corresponding liver lipid concentration (biochemistry data) or percent fractional area (histology data), respectively.
Hematoxylin eosin
Manufactured by Agilent Technologies
Sourced in Denmark
Hematoxylin-eosin is a common staining method used in histology and pathology to visualize the structure of cells and tissues. It utilizes two dyes, hematoxylin and eosin, to stain the nuclei and cytoplasm, respectively, providing a contrast that enables the examination of cellular and tissue morphology.
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Lab products found in correlation
Anti galectin 3, by BioLegend (2 mentions)
Picrosirius red, by Merck Group (1 mentions)
Anti type 1 collagen col1a1, by Southern Biotech (1 mentions)
Prism v7, by GraphPad (1 mentions)
Visiomorph, by Visiopharm (1 mentions)
Anti type 1 collagen, by Merck Group (1 mentions)
Nanozoomer s210 digital slide scanner, by Hamamatsu Photonics (1 mentions)
3 protocols using hematoxylin eosin
1
Comprehensive Liver Analyses in NAFLD
2
Histological Evaluation of Liver Biopsies
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Liver biopsies were collected from the left lateral lobe and fixed overnight in 4% paraformaldehyde. Liver biopsies were paraffin embedded, sectioned and stained with Hematoxylin & Eosin (Dako, Glostrup, Denmark), Pico-Sirius red (PSR, Sigma-Aldrich, Brøndby, Denmark), anti-type-1-collagen (Sourthern Biotech), anti-alfa-SMA (Abcam, Cambridge, UK) or anti-Galectin-3 (Biolegend, San Diego, CA) as described previously36 (link),37 (link). The NAS and fibrosis staging system outlined by Kleiner et al.6 (link) was applied by experienced histopathologists to score liver biopsies for steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis. Quantitative image analysis of stained liver sections were analyzed using the digital imaging software (Visiomorph; Visiopharm, Hørsholm, Denmark)36 (link),37 (link). Histo-chemical positive staining areas were expressed relative (%) to the total tissue sectional area (fractional area). Quantitative histological data were analyzed using GraphPad Prism v.7.04 software (GraphPad, LaJolla, CA), and results are shown as mean of n = 4–5 ± SEM. A students t-test was performed for quantitative histology data. All histological assessments were performed by a expert pathologist blinded to the experimental groups.
3
Histological Evaluation of NASH Models
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For the CDAA efficacy study, liver tissue was fixed in 10% neutral-buffered formalin for 24 h and paraffin embedded for routine processing. Liver sections cut from paraffin blocks were stained with H&E for blinded scoring of macrovesicular and microvesicular steatosis, inflammation and hepatocyte ballooning as previously described (Kleiner et al., 2005) . Blinded scoring was performed by an experienced pathologist at UCSD (San Diego, CA, USA).
Immunohistochemistry for MPO and alpha-smooth muscle actin (α-SMA), TUNEL staining, and PSR staining were performed as described previously (Wree et al., 2014b) . Stained liver sections were quantitated using bright field images captured with NanoZoomer S210 Digital Slide Scanner (Hamamatsu, Iwata City, Japan). For the DIO model of NASH, paraformaldehyde-fixed liver pre-and post-biopsies were paraffin-embedded, sectioned and stained with hematoxylineosin (Dako, Glostrup, Denmark), Pircrosirius red (Sigma-Aldrich, Broendby, Denmark), antitype I Collagen (cat. no. 1310-01, SouthernBiotech, Birmingham, AL, USA), and anti-MPO (cat.
no. RB-373-A1, ThermoFisher) as described previously (Tolbol et al., 2018) .
Immunohistochemistry for MPO and alpha-smooth muscle actin (α-SMA), TUNEL staining, and PSR staining were performed as described previously (Wree et al., 2014b) . Stained liver sections were quantitated using bright field images captured with NanoZoomer S210 Digital Slide Scanner (Hamamatsu, Iwata City, Japan). For the DIO model of NASH, paraformaldehyde-fixed liver pre-and post-biopsies were paraffin-embedded, sectioned and stained with hematoxylineosin (Dako, Glostrup, Denmark), Pircrosirius red (Sigma-Aldrich, Broendby, Denmark), antitype I Collagen (cat. no. 1310-01, SouthernBiotech, Birmingham, AL, USA), and anti-MPO (cat.
no. RB-373-A1, ThermoFisher) as described previously (Tolbol et al., 2018) .
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