Oil Red O and Sirius Red staining

For Oil Red O staining, the frozen sections were fixed with 10% formalin for 10 min, placed in 60% isopropyl alcohol and stained in 0.5% Oil Red O solution for 10 min. The slides were transferred to 60% isopropyl alcohol, rinsed in distilled water and processed for hematoxylin counter staining. For Sirius Red staining, the sections were fixed with 10% formalin for 10 min, incubated with a 0.1% Sirius Red solution dissolved in aqueous saturated picric acid for 1 h, washed in acidified water (0.5% acetic acid), dehydrated, and mounted on slides. Photomicrographs of tissue sections were taken using an Olympus BX53 digital microscope and the positively stained area was quantified with ImageJ (NIH, Bethesda, MD) and presented as percent of total area of the examined field.