Formalin-fixed, paraffin-embedded liver and adipose tissue sections were stained using Hematoxylin and Eosin (H&E). A semi-quantitative scoring system was used to assess hepatocyte steatosis by investigators blinded to the study (0, 1-5% of total area; 1, 5 – 33% of total area; 2, 33 – 66% of total area; 3, >66% of total area [40 (link)]. Adipocyte diameter and surface area were calculated using Adiposoft- ImageJ software.
Oil red O staining of liver sections was performed as described earlier [41 ]. Briefly, cryosections (6-8 microns in thickness) were air-dried and fixed in buffered formalin for 10 min. Sections were washed with water, rinsed in 60% isopropanol and stained with 0.3% Oil Red O solution for 15 min. After rinsing with 60% isopropanol, sections were stained with haematoxylin for 2 min, rinsed in water and mounted using an aqueous mounting media. Photographs were taken using Zeiss Axioplan imaging microscope at 20X magnification.
Oil red O staining of liver sections was performed as described earlier [41 ]. Briefly, cryosections (6-8 microns in thickness) were air-dried and fixed in buffered formalin for 10 min. Sections were washed with water, rinsed in 60% isopropanol and stained with 0.3% Oil Red O solution for 15 min. After rinsing with 60% isopropanol, sections were stained with haematoxylin for 2 min, rinsed in water and mounted using an aqueous mounting media. Photographs were taken using Zeiss Axioplan imaging microscope at 20X magnification.
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