Eclipse 90i microscope

Imaging of tissue sections stained by immunofluorescence or Masson trichrome was done using a Nikon Eclipse 90i microscope and the NIS Elements 3.2 software. Images were analyzed using the Image J 1.45s software (National Institute of Health, Bethesda, MD, USA).

Paraffin-embedded tissues were sectioned to a thickness of 4–5 μm and fixed to glass slides. Slides were deparaffinised and stained with hematoxylin-eosin. The severity of steatosis was determined in100 hepatocytes of two liver tissue sections per mouse and scored as follows: grade 0 when fat was not detected in hepatocytes; grade 1 when fat occupied less than 30% of hepatocytes; grade 2 when fat occupied between 30 and 60% of hepatocytes; grade 3, when fat occupied more than 60% of hepatocytes as described previously [17 (link)].
Adipocyte cell sizes were measured in 100 cells of two sections of epididymal adipose tissue per mouse and adipocyte cell sizes were expressed as area ranges using the following ranges: <2000, 2000–4000, 4000–6000 and 6000–7000 μm², as described previously [17 (link)]. All parameters were measured in a NIKON Eclipse 90i Microscope, using the NIS Elements BR 2.3 basic research software (Kingston, Surrey, KT25PR, England). All histological analyses were conducted blind by an experienced histologist.