Summary
Filipin is used for ultrastructural cytochemical localization of cholesterol in biological membranes. It binds to unesterified 3β-hydroxy-sterols forming 25 nm complexes which are readily recognized in freeze-fracture replicas. Since most investigations with filipin have been performed in isolated cells (tissue culture, cell suspensions etc.) we have investigated the conditions for reproducible labeling of cholesterol in membranes of parenchymatous organs. Vibratome sections of rat kidney fixed by glutaraldehyde perfusion were incubated in filipin and freeze-fracture replicas were prepared using standard techniques. The concentration of filipin, the thickness of vibratome sections and the incubation time and temperature were varied over a wide range. Optimal results were obtained with 50 μm thick tissue slices incubated in 400 μg/ml of filipin for 46 h at room temperature. Under these conditions lysosomes were consistently labeled while mitochondria and the endoplasmatic reticulum were negative. Peroxisomes showed a little or no labeling at all while the nuclear envelope was heavily labeled in some cells being negative in others. The method described here should be useful in investigation of the role of cholesterol in function of biological membranes in parenchymatous organs and compact tissues.
Similar content being viewed by others
References
Alecio MR, Golan DE, Veatch WR, Rando RR (1982) Use of fluorescent cholesterol derivative to measure lateral mobility of cholesterol in membranes. Proc Natl Acad Sci USA 79:5171–5174
Andersson Forsmann C (1985) Freeze-fracture cytochemistry of sympathetic ganglia. Distribution of filipin and tomatin induced membrane deformations in neurons and satellite cell. Histochemistry 82:209–218
Andrews LD, Cohen AJ (1983) Freeze-fracture studies of photoreceptor membranes: New observations bearing upon the distribution of cholesterol. J Cell Biol 97:749–755
Bittmann R (1978) Sterol-polyene antiboitic complexation: Probe of membrane structure. Lipids 13:686–691
Bloch KE (1983) Sterol structure and membrane function. Crit Rev Biochem 14:47–92
Branton D, Bullivant S, Gilula NB, Packer L, Karnovsky MJ, Moor H, Mühlethaler K, Northcote DH, Satir B, Speth V, Staehlin LA, Steere RL, Weinstein RS (1975) Freeze-etching nomenclature. Science 190:54–56
Chabanel A, Flamm M, Sung KLP, Lee MM, Schachter D, Chien S (1983) Influence of cholesterol content on red cell membrane viscoelasticity and fluidity. Biophys J 44:171–176
Chailley B, Boisvieux-Ulrich E (1985) Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in Quail oviduct. J Histochem Cytochem 33:1–10
De Kruijff B, Demel RA (1974) Polyene-sterol interactions in membranes of Acholesplasma laidlawii cells and lecithin membranes. III. Molecular structure of the polyene antibiotic-cholesterol complexes. Biochim Biophys Acta 339:57–70
Demel RA, De Kruijff B (1976) The function of sterols in membranes. Biochim Biophys Acta 457:109–132
Elias PM, Goerke J, Fried DS (1978) Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes. J Cell Biol 78:577–596
Elias PM, Friend DS, Goerke J (1979) Membrane sterol heterogenity. Freeze-fracture detection with saponins and filipin. J Histochem Cytochem 27:1247–1260
Fahimi HD, Kalmbach P, Stegmeier K, Stork H (1980) Comparison between the effects of Clofibrate and Bezafibrate upon the ultrastructure of at heart and liver. In: Greten H, Lang PD, Schettler G (eds) Lipoproteins and coronary heart disease. New aspects in the diagnosis and therapy of disorders of lipid metabolism. Gerhard Witzstrock, New York, pp 64–75
Friend DS (1980) Freeze-fracture alterations in guinea pig sperm membranes preceding gamete fusion. In: Gilula MB (ed) Membrane — membrane interactions. Raven Press, New York, pp 153–165
Friend DS, Bearer EL (1981) Beta-hydroxysterol distribution as determined by freeze-fracture cytochemistry. Histochem J 13:535–546
Fujiki Y, Fowler S, Shio H, Hubbard AL, Lazarow PB (1982) Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: Comparison with endoplasmic reticulum and mitochondria membranes. J Cell Biol 93:103–110
Fujimoto T, Ogawa K (1983) Uneven formation of filipin-sterol complexes in frog urinary bladder epithelium. Acta Histochem Cytochem 16:513–521
Gebhardt R (1983) Primary cultures of rat hepatocytes as a model system of canalicular development, biliary secretion and intrahepatic cholestasis. III. Properties of the biliary transport of IgA revealed by immunofluorescence. Gastroenterology 84:1462–1471
Henning R, Kaulen HD, Stoffel W (1970) Isolation and chemical composition of the lysosomal and the plasma membrane of the rat liver cell. Hoppe-Seyler's Z Physiol Chem 351:1191–1199
Hüttner J, Walker C, Gabbiani G (1985) Aortic endothelial cell during regeneration. Remodeling of cell junctions, stress fibers and stress fiber-membrane attachment domains. Lab Invest 53:287–302
Jain MK (1975) Role of cholesterol in biomembranes and related systems. Curr Top Membr Transp 6:1–57
Kalmbach P, Fahimi HD (1978) Peroxisomes: Identification in freeze-etch preparations of rat kidney. Cell Biol Int Rep 2:389–396
Karnovsky MJ, Kleinfeld AM, Hoover RL, Klausner RD (1982) The concept of lipid domains in membranes. J Cell Biol 94:1–6
Kim J, Okada Y (1983) Asymetric distribution and temperaturedependent clustering of filipin-sterol complexes in the nuclear membrane of Ehrlich ascites tumor cells. Eur J Cell Biol 25:131–135
Kinsky SC, Luse SA, Van Deenen LA (1966) Interaction of polyene antibiotics with natural and artificial membrane systems. Fed Proc 25:1503–1510
Korn ED (1969) Cell membranes: structure and synthesis. Annu Rev Biochem 38:263–322
Kruth HS, Blanchette-Mackie J, Avigan J, Gamble W, Vaughan M (1982) Subcellular localization and quantification of cholesterol in cultured human fibroblasts exposed to human low density lipoprotein. J Lipid Res 23:1128–1135
Madara JL, Bye WA, Trier JS (1984) Structural features of and cholesterol distribution in M-cell membranes in guinea pig, rat and mouse Peyer's patches. Gastroenterology 87:1091–1103
Matsuda H, Fujita H, Ishimura K (1983) Freeze-fracture images of distribution of filipin-cholesterol complexes in the oviduct epithelium of mice. Acta Histochem Cytochem 16:112–118
McGookey DJ, Fagerberg K, Anderson RGW (1983) Filipin-cholesterol complexes form in uncoated vesicle membrane derived from coated vesicles during receptor-mediated endocytosis of low density lipoprotein. J Cell Biol 96:1273–1278
Miller RG (1984) The use and abuse of filipin to localize cholesterol in membranes. Cell Biol Int Rep 8:519–535
Montesano R, Perrelet A, Vassalli P, Orci L (1979) Absence of filipin-sterol complexes from large coated pits on the surface of culture cells. Proc Natl Acad Sci USA 76:6391–6395
Montesano R, Vassalli P, Perrelet A, Orci L (1980) Distribution of filipin-cholesterol complexes at sites of exocytosis — a freezefracture study of degranulating mast cells. Cell Biol Int Rep 4:975–984
Norman AW, Demel RA, De Kruijff B, Van Deenen LLW (1972) Studies on the biological properties of polyene antibiotics: evidence for the direct interaction of filipin with cholesterol. J Biol Chem 247:1918–1929
Orci L, Montesano R, Meda P, Malaisse-Lagae F, Brown D, Perrelet A, Vassalli P (1981a) Heterogeneous distribution of filipincholesterol complexes across the cisterna of the Golgi apparatus. Proc Natl Acad Sci USA 78:293–297
Orci L, Singh A, Amherdt M, Brown D, Perrelet A (1981b) Microheterogeneity of protein and sterol content in kidney podocyte membran. Nature 293:646–647
Orci L, Brown D (1983) Distribution of filipin-sterol complexes in plasma membranes of the kidney. II. The thin limbs of Henlcy's Loop. Lab Invest 48:80–89
Pelletier RM, Friend DS (1983) The sertoli cell junctional complex: structure and permeability to filipin in the neonatal and adult Guinea pig. Am J Anat 168:213–228
Robenek H, Greven H (1981) Freeze-fracture evidence for high cholesterol content in nuclear membranes of a larval urodelan epidermis. Eur J Cell Biol 25:131–135
Robenek H, Jung W, Gebhardt R (1982) The topography of filipincholesterol complexes in the plasma membrane of cultured hepatocytes and their relation to cell junction formation. J Ultrastruct Res 78:95–106
Robinson JM, Karnovsky MJ (1980) Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol. J Histochem Cytochem 28:161–168
Severs NJ (1981) Localization of cholesterol in the golgi apparatus of cardiac muscle cells. Experientia 37:1195–1198
Severs NJ, Robenek H (1983) Detection of microdomains in biomembranes. An appraisal of recent developments in freeze-fracture cytochemistry. Biochim Biophys Acta 737:373–408
Severs NJ, Simons HL (1983) Failure of filipin to detect cholesterol-rich domains in smooth muscle plasma membrane. Nature 303:637–638
Simionescu N, Lupu F, Simionescu M (1983) Rings of membrane sterols surround the openings of vesicles and fenestrae in capillary endothelium. J Cell Biol 97:1592–1600
Stetson DL, Wade JB (1983) Ultrastructural characterization of cholesterol distribution in toad bladder using filipin. J Membr Biol 74:131–138
Trier JS, Madara JL (1984) Distribution of filipin-sterol complexes in villus goblet cell membrane of rat small intestine. Lab Invest 50:673–682
Verkleij AJ, De Kruijff B, Gerritsen WF, Demel RA, Van Deenen LLM, Ververgaert PHJ (1973) Freeze-etch electron microscopy of erythrocytes, Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B. Biochim Biophys Acta 291:577–581
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ginsbach, C., Fahimi, H.D. Labeling of cholesterol with filipin in cellular membranes of parenchymatous organs. Histochemistry 86, 241–248 (1987). https://doi.org/10.1007/BF00490254
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00490254