(a) Representative photomicrograph (original magnification ×40) showing that the vacuoles (arrow) are negative for Oil Red O Staining, (b) Representative photomicrograph illustrating cholesteryl esters with fluorescent dye Nile red staining in HFD (arrows), (c, d) Representative photomicrographs (original magnification ×1000) of semi-thin sections (original magnification ×1000) illustrating Toluidine Blue–positive dark blue vacuoles (arrow). Electron microscopy evaluation of ultrastructure of vacuoles in proximal tubules in mice treated with a HFD: presence of (e, f) enlarged clear vacuoles and (g, h) multilaminar inclusions (i: higher magnification), (j, k) Representative photomicrographs (original magnification ×1000) illustrating phospholipid accumulation in vacuolated tubule with Luxol Fast Blue in HFD (arrow, vacuoles stained in blue).