Quantifying Hepatic Steatosis and Inflammation

Liver tissue was harvested at ZT 6 (12 p.m.) following fasting from ZT 0 (6 a.m.) on the final day of the experiment. Hepatocellular steatosis was measured by Oil Red O staining on 7 μm cryosections of liver tissue [30 (link)]. Images were scanned by bright-field light microscopy (Eclipse TE300, Nikon, NY, USA) at ×20 magnification. The area of Oil Red O lipid staining was measured using Image J software (Version 1.54g) [47 (link)]. Hepatic steatosis was further assessed by hematoxylin and eosin (H&E) staining of 5 μm liver tissue sections of paraffin-embedded liver tissue. Images were scanned by a Nanozoomer 2.0HT Slide Scanner (Hamamatsu Photonics, Bridgewater, NJ, USA), and the number and area of lipid droplets (stained negative for H&E) were measured using Image J. Hepatic inflammation was assessed by counting infiltrating immune cells in the H&E-stained sections. Inflammation scoring was performed by a trained pathologist blinded to the sample identities. Liver fibrosis was assessed by Sirius Red staining and quantified by Image J.

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Das M., Kumar D., Sauceda C., Oberg A., Ellies L.G., Zeng L., Jih L.J., Newton I.G, & Webster N.J. (2024). Time-Restricted Feeding Attenuates Metabolic Dysfunction-Associated Steatohepatitis and Hepatocellular Carcinoma in Obese Male Mice. Cancers, 16(8), 1513.

Publication 2024

Eclipse TE300 Nanozoomer 2.0HT Slide Scanner

Corresponding Organization : University of California, San Diego

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Variable analysis

independent variables

Time of tissue harvesting (ZT 6, 12 p.m.)
Fasting duration (from ZT 0, 6 a.m. on the final day of the experiment)

dependent variables

Hepatocellular steatosis measured by Oil Red O staining
Number and area of lipid droplets measured by H&E staining
Hepatic inflammation assessed by counting infiltrating immune cells in H&E-stained sections
Liver fibrosis assessed by Sirius Red staining and quantified by Image J

control variables

Cryosection thickness (7 μm)
Paraffin-embedded liver tissue section thickness (5 μm)
Microscope magnification (×20)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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