ARTICLE |
Correspondence to: Nalini M. Rajamannan, Northwestern U. Medical School, 675 N St Clair St. Suite 10-240, Chicago, IL 60611. E-mail: n-rajamannan@northwestern.edu
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Summary |
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Ultrastructural analysis of aortic valve endothelial cells subjected to growth arrest revealed many vesicles defined as caveolae by the localization of caveolin. Translocation of caveolin after exposure to oxidized LDL suggests that the localization of caveolin may be a valuable tool to study models of early atherogenesis. In this study, several antigen retrieval protocols were tested in osmium-fixed and Spurr-embedded cells to determine the optimal method of antigen retrieval in our model system. SDS produced the most consistent labeling pattern. A quantitative evaluation revealed that SDS significantly increased the labeling density in Spurr-embedded cells. The labeling pattern appeared as clusters of gold particles, 1540 nm in diameter, that were associated with membranes of a similar size which may represent the neck region of the caveolae.
(J Histochem Cytochem 50:617627, 2002)
Key Words: antigen retrieval, caveolin 1, immunoelectron microscopy, aortic valve endothelial cells, caveolae, quantitation, plasmalemmal vesicles, gold labeling, ultrastructure, LR White, Spurr resin
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Introduction |
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AORTIC VALVULAR HEART DISEASE is the third most common indication for valve replacement in the United States. Whereas the etiology of the degeneration is not well understood, the histology of removed valves shows atherosclerotic lesions and calcification similar to the vascular changes seen in cardiac heart disease. The normal aortic valve is composed of collagen, elastin, and fibroblasts and has layers of endothelial cells on both surfaces between blood and the stroma (
On the luminal surface of normal vascular endothelial cells, the adherence of cationic ferritin indicates a cell surface that is negatively charged, except over the openings of the plasmalemmal vesicles (
Atherosclerosis is also linked to a decrease in endothelial nitric oxide (NO) production (
Caveolar structure has been studied extensively and caveolae are described as small flask-shaped vesicles limited by a trilaminar membranes that are often continuous with the cell plasmalemma, hence the name plasmalemmal vesicles (
Caveolin 1 has been labeled extensively for transmission electron microscopy using frozen sections (
Ultrastructural details are excellent when tissues are fixed in osmium tetroxide and embedded in epoxy resin, but etching is often necessary to increase the antigenicity. After etching with sodium metaperiodate or hydrogen peroxide, an increase in nonspecific labeling of caveolin 1 was reported in lung tissue (
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Materials and Methods |
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Reagent and Antibody Sources
The reagents for these experiments were obtained from the following commercial sources: PBS plus Tween 20 (PBS-T), sodium citrate, SDS, glycine, tannic acid, goat anti-rabbit IgG conjugated to horseradish peroxidase, and sodium metaperiodate were from Sigma Chemical (St Louis, MO). Rabbit anti-caveolin 1 and human endothelial cell lysate were from Transduction Laboratories (Franklin Lakes, NJ). Spurr resin embedding components, DER-736, ERL-4206, NSA, and DMAE, parlodion, glutaraldehyde, uranyl acetate, osmium tetroxide, nickel grids (300-mesh thin bar), lead citrate, and sodium cacodylate were from Electron Microscopy Sciences (Fort Washington, PA). Goat anti-rabbit conjugated to 10-nm and 5-nm colloidal gold, ECL Western blotting detection reagents, and normal goat serum were from Amersham Life Sciences (Arlington Heights, IL), acetone from JT Baker (Phillipsburg, NJ), amyl acetate from Mallinckrodt (Paris, KY), formaldehyde from Ted Pella (Redding,CA), Dulbecco's PBS without calcium or magnesium from BioWhittaker (Walkersville, MD), gels from BioRad (Hercules, CA), serum-free media, medium 199, and fetal bovine serum from Life Technologies (Rockville, MD).
Fixation and Embedding
Aortic valve endothelial cells (AVECs) were cultured from porcine valves after enzymatic digestion as previously described (
Normal AVECs were incubated for 24 hr in serum-free medium and fixed overnight in 4% formaldehyde plus 1% glutaraldehyde in phosphate buffer (
A duplicate set of serum-free cells was fixed overnight in 4% formaldehyde plus 0.2% glutaraldehyde in phosphate buffer. Cells were rinsed in phosphate buffer and dehydrated in a series of ethanols to 80% while progressively lowering the temperature to -20C. The cells were infiltrated in LR White resin at -20C overnight, embedded in fresh resin at room temperature (RT), and polymerized at 50C for 2 days. This embedding protocol has been used successfully to label collagen Type IV (
Sections with a silver interference color (80 nm thick) were mounted on 300-mesh nickel grids for labeling. Some grids were mounted on parlodion membranes before labeling to minimize section wrinkling. These membranes were made from 1.5% parlodion dissolved in 1:1 acetone:amyl acetate. Both grid and section were deposited on the dried parlodion films on the platform. Gold conjugate (5 nm) was dried onto a membrane and negatively stained with uranyl acetate. The diameter of the cloud around the dense particle was measured.
Antigen Retrieval
For a comparison of AR protocols, grids were exposed to sodium metaperiodate, heated citrate buffer, or a combination of sodium metaperiodate and heated citrate buffer. Sections were treated in a saturated aqueous solution of sodium metaperiodate for 10 minutes (
Immunoelectron Microscopy
The antibody to caveolin 1 was a rabbit polyclonal to the N-terminus of human caveolin 1 that included amino acid sequences 197. This antibody was diluted 1:50 in PBS-T. Blocking buffer was PBS with added 0.05% Tween-20 (PBS-T), 1% glycine, and 2% normal goat serum. The secondary antibody was goat anti-rabbit conjugated to 5- or 10-nm colloidal gold and was diluted 1:100 in PBS-T. After AR grids were incubated in blocking buffer for 15 min. The grids were incubated in primary antibody for 3 hr at RT. Grids were rinsed in four changes of PBS-T, incubated for 60 min in secondary goat anti-rabbit conjugated to 5-nm gold, rinsed in PBS-T, rinsed in water, and dried. Some sections were examined after staining with uranyl acetate and lead citrate and some were examined without any post-labeling staining. After AR in a combination of sodium metaperiodate and heated citrate buffer, controls were labeled with a nonspecific rabbit antibody and with the omission of primary antibody. Controls were also incubated in SDS and labeled without primary antibody.
Quantitative Methods
The minor diameter of all vesicle membranes was measured in 39 fields of 1 µm2 of Spurr-embedded cells subjected to SDS. One group of membranes surrounded dense vesicle matrix and the other group surrounded clear areas within the vesicle. When gold labeling density (gold particles/µm2) in the different embedding and antigen retrieval media was compared, the gold particles were counted in 25 fields. Within the gold clusters, the distance between the center of the gold particle to the center of the nearest neighboring gold particle was measured. All statistical comparisons were made with the two-tailed Student's t-test assuming unequal variances.
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Results |
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General Structural Appearance and Size
Porcine AVECs under normal culture conditions usually contain many mitochondria, Golgi apparatus, and an occasional group of caveolae (Fig 1A). When these cell cultures were growth-arrested by exposure to serum-free medium for 24 h before fixation, the ultrastructural examination revealed many caveolae (Fig 1C and Fig 1D) and an occasional vesiculo-vacuolar organelle (VVO) (Fig 1B). When sectioned at a right angle to the plasma or vacuolar membrane, single caveolae appeared as flask-shaped invaginations of the membranes (Fig 1D). In this orientation, the neck of the caveola opened either into the exterior of the cell or into a larger vacuole. When sectioned through the midline of the caveolae, the single vesicles were more circular and the vesicle matrix was either homogeneously dense or had a clear area within the dense matrix of the vesicle (Fig 1C). This clear area may represent the caveolar stomata, and in rare cases a dense central knob can be demonstrated within these clear areas (Fig 4B).
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When AVECs were processed in LR White for maximal antigenicity (Fig 2), a lack of membrane contrast in mitochondria, caveolae, and plasmalemma was apparent. The labeling pattern for caveolin was clustered and usually found near the plasmalemma (Fig 2), but assessing the specificity of the label was difficult when the trilaminar membrane structure was not well contrasted. LR White resin does not polymerize when osmium is used for postfixation. Without osmium and with partial dehydration, the trilaminar structure of the membranes appeared in reverse contrast with a dark middle layer and pale outer layers (Fig 2 inset, white arrow).
Antigen Retrieval
In Spurr-embedded cells, several AR methods were tested in the search for a protocol that would retain the membrane structure of the vesicles and increase the labeling density for caveolin. AR in sodium metaperiodate showed a good labeling pattern but produced a dense precipitate that was difficult to remove completely from the sections. A nonspecific labeling was often associated with this electron-dense debris. When heated citrate buffer was used as a single AR treatment, the specificity of the label appeared to be improved over sodium metaperiodate used alone. A combination of sodium metaperiodate followed by heated citrate buffer was effective in producing a specific caveolin label, but section wrinkling often obstructed an evaluation of the labeling pattern. The most consistent labeling pattern was found after AR in SDS, and caveolin was consistently localized over vesicle profiles with little contamination and no section wrinkling.
Labeling Pattern
In AVECs subjected to serum-free medium, the labeling of caveolin 1 was most often associated with membranes of caveolae. Signal was not found on every caveola and was not associated with the larger vacuoles or clathrin-coated pits. This pattern was similar in cells embedded in both LR White and Spurr resins, but the labeling density in Spurr resin was significantly decreased compared to the labeling density in LR White-embedded cells. Control cells labeled without primary antibody were negative with SDS AR.
After AR in SDS, caveolin labeling was demonstrated in AVECs that had been embedded in Spurr resin. The labeling was clustered and consistently localized over vesicle membranes. Fig 3 shows some typical labeling patterns found in Spurr-embedded cells. Whereas the label was found dispersed as single gold particles over a vesicle membrane (Fig 3A and Fig 3B), some clusters of gold particles were found over membranes surrounding dense areas of the vesicle (Fig 3C3E) However, most of the clusters were associated with membranes of smaller diameter that surrounded clear areas in the vesicle (Fig 3A3F). These gold clusters were not amorphous aggregations of gold but were often aligned linearly (Fig 3F) and separated by distances greater than 15 nm from the next nearest neighboring gold particle. In Fig 4B, a vesicle was sectioned through the dense knob and the radiating filamentous structures appeared similar to those published earlier by
Quantitative Comparisons
Fig 5 shows the distribution of two membrane populations that were measured in AVECs embedded in Spurr resin and labeled for caveolin after SDS AR. These membranes' profiles are parts of the same vesicles but they may represent two regions of the vesicle that can be separated by size. The diameters of vesicle membranes surrounding a dense matrix may represent the body of the vesicle, and the membranes surrounding a clear area within the vesicle probably represent the narrowing of the membranes in the neck region of the vesicle. Fig 5 suggests that these two vesicle regions on the same vesicles can be separated by size, and the two membrane groups are significantly different, as shown in Table 1. The two groups were compared statistically with the two-tailed Student's t-test assuming unequal variances.
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Because LR White is considered optimal for preserving antigenicity, the gold labeling density of caveolin was determined with and without SDS antigen retrieval. These densities were compared to gold labeling density in Spurr-embedded cells with and without SDS retrieval. The quantitative results are summarized in Table 2 and graphically represented in Fig 6.
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The density of gold particles/µm2 was significantly different when the two embedding media were compared (p<0.001), but the AR in SDS did not significantly increase the labeling efficiency in the LR White-embedded cells (p=0.763). In Spurr-embedded cells, a significant increase in labeling density could be demonstrated (p<0.001) when SDS was used as an AR medium. Given the nature of the structural data present in Spurr-embedded cells, an increase in labeling density after SDS treatment may be a valuable tool in our study of AVECs.
In Spurr-embedded cells with AR in SDS, many single gold particles were found over caveolae membranes and were isolated from their nearest neighboring gold particle by distances of over 100 nm. Gold particles that were closer than 100 nm were designated as gold clusters, and 61 clusters were identified that included 71% of the gold particles counted. Within the gold clusters, the distances between the particles and their nearest neighbor was measured and a distribution of those distances appears in Fig 7. The clusters are concentrated between 5 and 30 nm.
The cloud around the gold probe (5 nm) was negatively stained and the diameter of the "cloud" was measured in 172 particles. Table 3 summarizes the size of the negatively stained cloud around the 5-nm gold particle.
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If this cloud represents the distance at which gold can be aggregated, then gold particles closer than 15 nm will be, for this discussion, considered aggregated. Particles with interpoint distances greater than 15 nm will be considered as labeling associated with a clustered epitope on the section surface. Clustered labeling of caveolin is quite apparent at 1540 nm but is almost nonexistent at distances of 4080 nm. These data are not conclusive but support the theory that caveolin labeling is associated with the smaller membranes surrounding the clear areas within the vesicle (2040 nm) and suggest that these areas may represent the narrower neck of the caveolae.
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Discussion |
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After arresting cell growth with serum-free medium, a considerable number of caveolae present as single caveola, groups of caveolae, or VVOs were demonstrated in AVECs. In these cells, caveolin label is associated with membrane-limited clear zones that are smaller and often enclosed within the larger vesicle membrane that defines the vesicle matrix. The diameters of these smaller membranes (2040 nm) are similar to those stomata or vesicle openings (3050 nm) observed in freeze-fractured preparations (
Caveolin has been associated with the cytoplasmic side of the caveolar membrane, and ultrastructural studies of caveolae revealed four to six filaments arranged concentrically on the cytoplasmic surface. When this striated coat was disrupted, the labeling of caveolin was greatly increased (
Our data suggest that the postembedding labeling of caveolin may be dependent on the orientation of the caveolae within the section. When the neck regions of the caveolae are exposed at the section surface, the epitope has access to the labeling solutions and the caveolin is labeled. The exposure of the epitope at the section surface may not be the only factor that governs the positive labeling of caveolin. Perhaps attachments between the monomers or other ligands attached to caveolin complex must be dissociated before the antibody has access to the N-terminus of the caveolin molecule. SDS is commonly used to dissociate proteins, but the caveolin complex of 14 or 15 monomers is somewhat resistant to dissociation by SDS (
Several AR techniques other than SDS were also effective in localizing caveolin 1 in AVECs fixed in osmium tetroxide and embedded in Spurr resin. AR with heated citrate buffer alone and with a combination of sodium metaperiodate followed by heated citrate buffer were equally effective in increasing the labeling of caveolin 1. When many AR methods were compared on tissue embedded in Araldite, the best quantitative increases in label were produced by a combination of sodium metaperiodate and heated citrate buffer (
In Spurr-embedded AVECs, sodium metaperiodate used alone showed an increase in caveolin 1 labeling. However, an electron-dense contamination often obscured the structural detail and this precipitate was labeled nonspecifically by gold particles. This electron-dense precipitate may be osmium that was removed from the section by sodium metaperiodate. Others have reported that sodium metaperiodate is effective in removing the effects of osmium fixation (
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Acknowledgments |
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We wish to thank Dr Ann Dvorak for her helpful comments on the manuscript, and Jon Charlesworth of Mayo Foundation's Electron Microscopy Core Facility.
Received for publication March 6, 2001; accepted December 12, 2001.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amino K, Honda Y, Ide C, Fujimoto T (1997) Distribution of plasmalemmal Ca(2+)-pump and caveolin in the corneal epithelium during the wound healing process. Curr Eye Res 16:1088-1095[Medline]
Bendayan M, Zollinger M (1983) Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J Histochem Cytochem 31:101-109[Abstract]
Blair A, Shaul PW, Yahanna IS, Conrad PA, Smart EJ (1999) Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation. J Biol Chem 274:32512-32519
Brorson SH (1999) Fixative-dependent increase in immunogold labeling following antigen retrieval on acrylic and epoxy sections. Biotech Histochem 74:248-260[Medline]
Brown D, Lydon J, McLaughlin M, StuartTilley A, Tyszkowski R, Alper S (1996) Antigen retrieval in cryostat sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol 105:261-267[Medline]
Bruns RR, Palade GE (1968) Studies on blood capillaries: I General organization of blood capillaries in muscle. J Cell Biol 37:244-276
Cayette AJ, Palacino JJ, Horten K, Cohen RA (1994) Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler Thromb 14:753-759[Abstract]
Cooper T, Napolitano LM, Fitzgerald MJ, Moore KE, Daggett WM, Willman VL, Sonnenblick EH, Hanlon CR (1966) Structural basis of cardiac valve function. Arch Surg 93:767-771[Medline]
deWeerd WF, LeebLundberg LM (1997) Bradykinin sequesters B2 brandykinin receptors and the receptor-coupled Galpha subunits Galphaq and Galphai in caveolae in DDT1 MF-2 smooth muscle cells. J Biol Chem 272:17858-17866
Dolo V, Li R, Dillinger M, Flati S, Manela J, Taylor BJ, Pavan A, Ladisch S (2000) Enrichment and localization of ganglioside G(D3) and caveolin-1 in shed tumor cell membrane vesicles. Biochim Biophys Acta 1486:265-274[Medline]
Dvorak AM, Kohn S, Morgan ES, Fox P, Nagy JA, Dvorak HF (1996) The vesiculo-vacuolar organelle (VVO): a distinct endothelial cell structure that provides a transcellular pathway for macromolecular extravasation. J Leukocyte Biol 59:100-115[Abstract]
Feng D, Nagy J, Hipp J, Dvorak H, Dvorak A (1996) Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor. Histamine and serotonin. J Exp Med 183:1981-1986[Abstract]
Fujimoto T, Fujimoto K (1997) Metal sandwich method to quick-freeze monolayer cultured cells for freeze fracture. J Histochem Cytochem 45:595-598
Fujimoto T, Hagiwara H, Aoki T, Kogo H, Nomura R (1998) Caveolae: from a morphological point of view. J Electron Microsc 5:451-460
Golfert F, Kasper M, vanEys GJ, Funk RH (1997) Cytoskeletal characterization of arteriovenous epitheloid cells. Histochem Cell Biol 108:513-523[Medline]
Griffith OW, Stuehr DJ (1995) Nitric oxide synthases: properties and catalytic mechanisms. Annu Rev Physiol 57:707-736[Medline]
Haasemann M, Cartaud J, MullerEsterl W, Dunia I (1998) Agonist-induced redistribution of bradykinin B2 receptor in caveolae. J Cell Sci 111:917-928
Hann CR, Springett MJ, Johnson DH (2001) Antigen retrieval of basement membrane proteins from archival eye tissues. J Histochem Cytochem 49:475-482
Johnson CM, Fass DN (1983) Porcine cardiac valvular endothelial cells in culture. A relative deficiency of fibronectin synthesis in vitro. Lab Invest 49:589-598[Medline]
Ju H, Zou R, Venema RJ, Venema RC (1997) Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 272:18522-18525
Kasper M, Golfert F, Funk RH (1997) Immunoelectron microscopical characterization of the epithelioid type of smooth muscle cells in human glomus organs. Ultrastruct Pathol 21:425-430[Medline]
Luetterforst R, Stang E, Zorzi N, Carozzi A, Way M, Parton RG (1999) Molecular characterization of caveolin association with the Golgi complex: identification of a cis-Golgi targeting domain in the caveolin molecule. J Cell Biol 145:1443-1459
Lupu C, Goodwin CA, Westmuckett AD, Emeis JJ, Scully MF, Kakkar VV, Lupu F (1997) Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium. Arterioscler Thromb Vasc Biol 17:2964-2974
Lupu F, Simionescu M (1985) Organization of the intercellular junctions in the endothelium of cardiac valves. J Submicrosc Cytol 17:119-132[Medline]
Manduteanu I, Popov D, Radu A, Simionescu M (1988) Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin. J Mol Cell Cardiol 20:103-118[Medline]
McDowell EM, Trump BF (1976) Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100:405-414[Medline]
Michel J, Feron O, Sase K, Prabhakar P, Michel T (1997) Caveolin versus calmodulin: counterbalancing allosteric modulators of nitric oxide synthase. J Biol Chem 272:25907-25912
Michel T, Li GK, Busconi L (1993) Phosphorylation and subcellular translocation of endothelial nitric-oxide synthase. Proc Natl Acad Sci USA 90:6252-6256[Abstract]
Murata M, Peranen J, Schreiner R, Weiland F, Kurzchalia TV, Simons K (1995) VIP21/Caveolin 1 is a cholesterol-binding protein. Proc Natl Acad Sci USA 92:10339-10343[Abstract]
Newmann GR, Campbell L, von Ruhland C, Jasani B, Gumbleton M (1999) Caveolin 1 and its cellular and subcellular immunolocalization in lung alveolar epithelium: implications for alveolar epithelial type I cell function. Cell Tissue Res 295:111-120[Medline]
Palade GE (1953) Fine structure of blood capillaries. J Appl Phys 24:1424
Palade GE, Bruns RR (1968) Structural modulations of plasmalemma vesicles. J Cell Biol 37:633-649[Medline]
Palmer RMJ, Ferrige AG, Mondada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524-526[Medline]
Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG (1992) Caveolin, a protein component of caveloae membrane coats. Cell 68:673-682[Medline]
Rothberg KG, Ying YS, Kamen BA, Anderson GW (1990) Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J Cell Biol 111:2931-2938[Abstract]
Sargiacomo M, Scherer PE, Tang ZL, Kubler E, Song KS, Sanders MC, Lisanti MP (1995) Oligomeric structure of caveolin 1: implications for caveolae membrane organization. Proc Natl Acad Sci USA 92:9407-9411[Abstract]
Sarphie TG (1985) Anionic surface properties of aortic and mitral valve endothelium from New Zealand White rabbits. Am J Anat 174:145-160[Medline]
Sarphie TG (1986) A cytochemical study of the surface properties of aortic and mitral valve endothelium from hypercholesterolemic rabbits. Exp Mol Pathol 44:281-296[Medline]
Sarphie TG (1987) Interactions of IgG and B-VLDL with aortic valve endothelium from hypercholesterolemic rabbits. Atherosclerosis 68:199-212[Medline]
Schaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna I, Ying Y, Anderson RGW, Michel T (1996) Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem 271:6518-6522
Scherer PE, Lewis RY, Volonte D, Engelman JA, Galbiati F, Couet J, Kohtz DS, vanDonselaar E, Peters P, Lisanti MP (1997) Cell-type and tissue-specific expression of caveolin-2. Caveolins 1(and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J Biol Chem 272):29337-29346
Schwab W, Kasper M, Gavlik JM, Schulze E, Funk RH, Shakbaei M (2000) Characterization of caveolins from human knee joint cartilage: expression of caveolin-1, -2 and -3 in chondrocytes and association with integrin beta 1. Histochem Cell Biol 113:221-225[Medline]
Severs NJ, Simons HL (1986) Caveolar bands and the effects of sterol-binding agents in vascular smooth muscle plasma membrane. Lab Invest 55:295-307[Medline]
Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem 41:1599-1604
Simionescu N, Simionescu M, Palade GE (1981) Differentiated microdomains on the luminal surface of the capillary endothelium: I Preferential distribution of anionic sites. J Cell Biol 90:605-613[Abstract]
Simionescu N, Vasile E, Lupu F, Popescu G, Simionescu M (1986) Prelesional events in atherogenesis: accumulation of extracellular cholesterol-rich liposomes in the intima and cardiac valves of hyperlipidemic rabbits. Am J Pathol 123:109-125[Abstract]
Song KS, Tang ZL, Li S, Lisanti MP (1997) Mutational analysis of the properties of caveolin 1. J Biol Chem 272:4398-4403
Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31-43[Medline]
Stahlhut M, Van Deurs B (2000) Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1 associated membrane domains by the actin cytoskeleton. Mol Biol Cell 11:325-337
Stang E, Kartenbeck J, Parton RG (1997) Major histocompatibility complex class I molecules mediate association of SV40 with caveolae. Mol Biol Cell 8:47-57[Abstract]
Stirling JW, Graff PS (1995) Antigen unmasking for immunoelectron microsocpy: labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium. J Histochem Cytochem 43:115-123
Takayama I, Terada N, Baba T, Ueda H, Kato Y, Fujii Y, Ohno S (1999) "In vivo cryotechnique" in combination with replica immunoelectron microscopy for caveolin in smooth muscle cells. Histochem Cell Biol 112:443-445[Medline]
Thyberg J (2000) Differences in caveolae dynamics in vascular smooth muscle cells of different phenotypes. Lab Invest 80:915-929[Medline]
Vane JR, Anggard EE, Botting RM (1990) Regulatory functions of the vascular endothelium. N Engl J Med 323:27-36[Medline]
Vasile E, Qu-Hong, Dvorak HF, Dvorak AM (1999) Caveolae and vesiculo-vacuolar organelles in bovine capillary endothelial cells cultured with VPF/VEGF on floating Matrigel-collagen gels. J Histochem Cytochem 47:159-168
Vasile E, Simionescu M, Simionescu N (1983) Visualization of the binding, endocytosis, and transcytosis of low-density lipoprotein in the arterial endothelium in situ. J Cell Biol 96:1677-1689[Abstract]
Westermann M, Leutbecher H, Meyer HW (1999) Membrane structure of caveolae and isolated caveolin 1-rich vesicles. Histochem Cell Biol 111:71-81[Medline]
Wu M, Fan J, Gunning W, Ratnam M (1997) Clustering of GPI-anchored folate receptor independent of both cross-linking and association with caveolin. J Membr Biol 159:137-147[Medline]