Nile Blue Is a Basic Dye of the Oxazine Group, Which Has Been Used in Several Microscopic and Histochemical Techniques (4, 6)

ACTA HISTOCHEM. CYTOCHEM. Vol. 16, No. 3, 1983

LETTER TO THE EDITOR

NILE BLUE SULFATE STAINING FOR DEMONSTRATION OF LIPIDS IN FLUORESCENCE MICROSCOPY

Nile blue is a basic dye of the oxazine group, which has been used in several microscopic and histochemical techniques (4, 6) . A staining method based on application of Nile blue sulfate was introduced early for the cytochemical demonstration of lipids (3, 8, 9), showing neutral fats and fatty acids in red and blue, respectively. There is evidence (1, 10) that aqueous solutions of Nile blue sulfate contain the blue cation of the dye, a red oxidation product (Nile red), and the orange-red imino base. According to this view, phospholipids and fatty acids stain blue because they react with the Nile blue cation, meanwhile neutral fats appear in red color. Other observations seem to be in disagreement with this staining mechanism (5, 6) . A fluorescence reaction in tissues after staining with Nile blue sulfate and brilliant cresyl blue has been found by Bozzo and Campos Vidal (2). During the course of investigations by using oxazine dyes we have observed that Nile blue sulfate produces a strong fluorescence reaction in lipid droplets. The fat body of Drosophila larvae is a continuous tissue mass which has been chosen as test material because of its abundance in lipidic inclusions. Drosophila hydei testes, surrounded by the fat body, were fixed in 5 % formaldehyde for 1-24 hr, washed, and then immersed in 0.1 mg/ml aqueous Nile blue sulfate (Fluka) for 30 min. After staining, the material was briefly washed and mounted with a drop of water. Procedures for lipid extraction were carried out before staining by using acetone or methanol/chloroform (1 : 2, v/v) at room temperature for 24 hr, followed by dehydration in ethanol series. In addition, smears of Ehrlich ascites tumor cells were fixed in 5 % formaldehyde for 15 min, stained with 0.1 mg/ml Nile blue sulfate solution for 8-20 mmn and mounted in water. Preparations were observed in a Zeiss Photomicroscope III equipped with an epifluorescence condenser RS and the filter set for green (546 nm) exciting light. After Nile blue staining, both the larval testes and Ehrlich cells show a blue or violet-blue color under bright field illumination (Fig. lA). The fat body and some vacuole-like areas in the cytoplasm of Ehrlich cells appear either unstained or very slightly stained in yellow-pink. When observed under fluorescence microscopy, the same preparations show a quite distinct and definite pattern, lipid droplets being the only structures which are highly fluorescent (Fig. 1B). A strong red emission is seen in a number of fat drops which escape from the disrupted fat body. Likewise, round lipidic inclusions of variable size in the cytoplasm of Ehrlich cells (7), show a bright red fluorescence (Table 1). Previous treatment with acetone or methanol/chloroform abolishes the fluorescence pattern by Nile blue, which indicates that lipids are the responsible components for this reaction. No fluorescence was observed in preparations which were not subjected to the staining solution. It is known that Nile blue forms a fluorescent red oxazone (Nile red) and a non- fluorescent orange-red base (1, 6, 10). Nile red is fat soluble (6) and could be the compound which accounts for the fluorescence reaction of lipids. The method de- scribed here has similarities with that of the benzpyrene-caffeine (6). However,

286 FLUORESCENCE OF LIPIDS BY NILE BLUE 287

Fies. IA, B Photomicrograph of a D. hydei testis surrounded by the fat body, after staining with Nile blue sulfate. A. Observed under bright field illumination. B. Observed under epifluorescence microscopy. x 43

TABLE 1. Staining and fluorescencereactions of Ehrlich ascites tumor cells after treatment with 0.1 mg/ml Nile blue sulfate for 8 min. 1HF and - indicate intense and no reaction, respectively.

1) Green exciting light was used. the use of Nile blue solutions lacks the disadvantages of the benzpyrene method, such as solubilization, reactivity and carcinogenic risk of the hydrocarbon. The technique we have presented is simple and reproducible, allowing to demonstrate 288 CANETE ET AL. lipidic inclusions with a high degree of resolution and brightness. Further studies on the application of Nile blue sulfate in fluorescence microscopy are in progress.

ACKNOWLEDGEMENTS This work was partially supported by a grant (4142-79) from the Comision Asesora de Investigacion Cientifica y Tecnica, Spain.

REFERENCES

1. Baker, J. R.: Principles of Biological Microtechnique. John Wiley and Sons, New York, 1958. 2. Bozzo, L., and Campos Vidal, B.: Nile blue sulphate and brilliant cresyl blue in fluorescence methods for the histochemical demonstration of lipids. Ann. Histochem. 13; 177, 1968. 3. Cain, A. J.: The use of Nile blue in the examination of lipoids. Quart. J. Microsc. Sci. 88; 383, 1947. 4. Gurr, E.: Synthetic Dyes in Biology, Medicine and Chemistry. Acad. Press, London and New York, 1971. 5. Lillie, R. D.: The mechanism of Nile blue staining of lipofuchsins. J. Histochem. Cytochem.4; 377, 1956. 6. Lillie, R. D.: Conn's Biological Stains. The William and Wilkins Co., Baltimore, 1977. 7. Paweletz, N.: Elektronenmikroskopische Untersuchungen an Unterlinien des Ehrlich'schen Mause-Ascites Tumors. Cytobiologie4; 103, 1971. 8. Smith, J. L.: On the simultaneous staining of neutral fat and fatty acid by oxazine dyes. J. Pathol. Bacteriol. 12; 1, 1907. 9. Smith, J. L., and Mair, W.: Fats and lipoids in relation to methods of staining. Scand. Arch. Physiol. 25; 245, 1911. 10. Thorpe, J. F.: A reaction of certain colouring matters of the oxazine series. J. Chem. Soc. 91; 325, 1907.

October 19, 1982 and in revised form Jaunary 14, 1983

M. CANETE,M. J. HAZENAND J. C. STOCKERT Departamentode Citologiae Histologia, Facultad de Ciencias UniversidadAutonoma de Madrid, Spain