The mechanism of Nile blue staining of lipofuscins.

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In 1908 J. L. Smith (23) reported simultaneous blue staining of fatty acids and red staining of neutral fats by aqueous solutions of the blue oxazine dye Nile blue sulfate (C.I. No. 913) and its red, oil soluble oxazone. In vitro tests indicated that shaking dilute Nile blue solutions with oleic acid imparted an intense blue color to the oil phase and decolorized the aqueous phase. Sulfuric acid was left in the aqueous phase and could be demonstrated with barium chloride. Olein was colored red by shaking with dilute Nile blue. The dye base imparted a red color to oils and fat solvents, which could be reextracted into dilute sulfuric acid, re-forming the blue salt. But the red oxazone, which imparted red color with intense yellow fluorescence to neutral oils, was not reextractible into dilute acid. This fluorescent red dye was formed more rapidly by boiling with dilute sulfuric acid and was identified by Thorpe (24) as an oxazone. He found that similar compounds were formed also by other phenonaphthoxazines. The 6-NH2, -NHR or -NR2 group was replaced by an =0, most rapidly with the unsubstituted -NH2. Smith explained the blue staining of the fatty acids by the formation of a blue fatty acid dye salt or “soap”, and stated that some time was required for the reaction, while the red coloration of liquid neutral fats occurred instantly by simple solution. The immediate blue coloration of oleic acid on shaking with aqueous Nile blue was ignored. This mechanism of staining of fatty acid by Nile blue has not been questioned since. Authors have either specifically agreed with it (2, 4, 5, 9, 19) or ignored the mechanisms (10, 12, 20, 21). Kaufmann and Lehmann (8) questioned the specificity of the reaction on the basis of elder pith model experiments. On rescanning their data it is seen that they reported blue staining of triolein and red colorations from mixtures of palmitic and stearic acids with triolein, findings not confirmed by Cain (2), and apparently not repeated by the authors, as the only discrepancies. Lison (13), however, regarded the coloration of fatty acids as quite unspecific, and as of the same nature as the staining obtained with many other basic dyes. Cain found it impossible to distinguish between Nile blue staining of proteins and of fatty acids, but found that only liquid fatty acids stained, and that palmitic and stearic acids would stain, if melted, just as Smith reported in 1908. Lennert (ii) found that all fatty acids which were liquid or soft at staining