The Use of Nile Blue Sulphate in the Histochemical Identification of Phospholipids

Representative extracted lipids were examined visually and spectrophoto-metrically following staining under varying conditions with the oxazine dye Nile blue sulphate. Optimum results in staining both extracted and tissue lipids were obtained with 1% acid-hydrolysed Nile blue prepared by boiling 200 ml of dye with 10 ml of 1% H2SO4 for 4 hr with refluxing. Staining was carried out at 70 C for 30 min. Under these staining conditions, extracted phospholipids were coloured intensely blue by the oxazine base of the dye, except for a synthetic cephalin which behaved as a neutral lipid. Neutral lipids (trigly-cerides and cholesteryl esters) were stained red by the oxazone derivative. Free fatty acids were coloured magneta-red due to a blend of red (oxazone) and blue (oxazine) staining. The procedure recommended clearly distinguished phospholipids from the other main lipid classes in tissue sections. Localisation of phospholipid was improved by also staining sections from which hydrophobic lipids had been removed by prior acetone extraction. Interpretation of both extracted and unextracted sections was restricted to clearly lipid material identified by parallel staining of sections with Sudan black.