Simultaneous demonstration of matrix lipid and proteoglycan in rat growth plate through selective preservation: A new technique

Abstract

The presence of lipid in cartilage has been determined through biochemical analysis and associated with mineralization (1,2,3), as is proteoglycan (4,5). However, unlike proteoglycan, an ultrastructural technique does not exist for cartilage lipid stabilization.Nile blue sulphate, a dye used in fluorescence microscopy to study lipids, when added to the initial glutaraldehyde fixation and followed by potassium ferrocyanide reduced osmium and en bloc, uranyl acetate, retained lipids in the cartilage matrix. In addition to the lipid stabilization, matrix proteoglycan was also rendered insoluable, two matrix components that have not been observed simultaneously before.Slices of growth plate cartilage from Sprague Dawley rats were fixed in 2% glutaraldehyde/0.1M Na phosphate (pH 7.4) containing 0.1% nile blue (min.2 hrs.), rinsed (15 min.) with 0.1M Na phosphate buffer containing 0.2M sucrose and 0.1% nile blue,post fixed with 1% aqueous OSO4 containing 1.5% potassium ferrocyanide, briefly washed in 0.1M Na acetate buffer (pH 6.3) then stained with 0.25% uranyl acetate in 0.1M Na acetate for one hour.