Immunologic Localization of Elastin by Electron Microscopy V. V. Damiano, A. Tsang, P. Christner, J. Rosenbloom, and G. Weinbaum
Research on the pathogenesis of experimental emphysema has involved studies of the distribution of and destruction of elastin in the alveolar interstitium. The ill-defined organization of elastin in the alveolar interstitium makes it difficult to identify the elastin specifically by staining procedures ordinarily used for electron microscopy. This problem becomes more significant when the elastic tissue is fragmented during emphysema development and localization of the elastin fragments is essential. Therefore, a specific technique using high-titer antibodies against purified canine lung elastin was developed. The primary antibody was used on preembedded or etched postembedded sections. Localization of the antielastin IgG was accomplished with ferritin-labeled rabbit antisheep IgG as the secondary antibody. Treatment with the preimmune serum gave negligible ferritin background staining. The antielastin antibody did not react with lung connective tissue proteins such as the microfibrillar component of elastin or collagen or proteoglycan. The antielastin antibody appeared to be species specific. The method may be useful for studies of experimental emphysema. (Am J Pathol 96:439456, 1979)
RECENT EMPHASIS in research on the etiology and pathogenesis of experimental pulmonary emphysema has been directed toward electron-microscopic studies of the distribution and destruction of elastin in the interstitial spaces of the alveolar walls. In order to accomplish such an analysis, specific electron-dense stains to visualize elastin in lung at the ultrastructural level have become increasingly important. With the use of standard electron-microscopic procedures involving primary fixation with glutaraldehyde and secondary fixation with osmium tetroxide, elastin is variably darkened, primarily because of the osmiophilic nature of elastin.1 Some control of the darkening effect may be achieved by control of the osmification time, but variability in the contrast ultimately depends upon the rate of penetration of osmium into the tissue. Early attempts to localize elastin by electron microscopy involved the use of phosphotungstic acid (PTA) (0. 1% in 50% ethanol).2 PTA combines vigorously with both elastin and collagen, but elastin is distinguished by the absence of a periodic structure characteristic of collagen. Since PTA From the Franklin Research Center, Philadelphia, Pennsylvania; the University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania; and the Albert Einstein Medical Center, Pulmonary Disease Section, Philadelphia, Pennsylvania. Supported by grants from the Council for Tobacco Research, Inc.-USA (Grant 901A) and the National Institutes of Health (Grants AM-02863, AM-02553, DE-02623, and PO1-HL-20994). Accepted for publication Marci 14, 1979. Address reprint requests to V. V. Damiano, PhD, Principal Scientist, Physics and Material Section, Franklin Research Center, Philadelphia, PA 19103. 0002-9440/79/0809-0439$01.00 439 O American Association of Pathologists
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extracts the reduced osmium from the thin sections, effective contrast, which is developed for other cellular components, is generally lost. Consequently, PTA is not effective in localizing elastin. Albert and Fleischer 3 developed a stain somewhat more specific for elastin by using silver salts of the sulfonated tetraphenol porphine (TPPS). They reported effective localization of elastin in both newborn and adult mouse aortas. More recently, a stain has been suggested for elastin, utilizing tannic acid-glutaraldehyde mixtures as the primary fixative. This was developed for the aorta, small arteries, cartilage, and bone connective tissue.4'5'6 A modification of this technique was used 7 by applying the tannic acid with uranyl acetate as a stain to postembedded (Epon 812) thin sections of chick aorta. All of the methods developed to date to localize elastin in tissue rely upon the imparting of an electron-dense stain of variable intensity and distribution to the elastin. Positive identification is based in part upon the relative association of the elastin with other components in the interstitial space and in part upon the contrast developed by the specific stain. Isolated elastin fragments encountered in emphysematous lung are difficult to identify positively on the basis of electron-dense contrast alone, since other osmiophilic components of the tissue, such as fibrin, chromatin, or lipids may develop similar electron-dense contrast and be mistaken for fragmented elastin. Immunologic staining procedures that utilize a well-defined marker such as ferritin offer the best potential method for locating specific cellular or tissue components,8'9"10 since the marker morphology and unique specificity as well as its electron density serve to specifically identify the component with which the antibody reacts. Immunologic stains, when applied directly to thin postembedded sections,"1"12 obviate the requirement of antibody penetration through cellular membranes or the dense interstitial space and offer the best approach to localization. Postembedding staining applied to serial sections further offers the possibility of applying different immunologic stains to sequential sections to localize more than one component in the same area. The objective of the present investigation was to develop an immunologic staining procedure that would specifically localize isolated elastin or elastin in the alveolar wall of postembedded thin sections prepared for electron microscopy. Once such a technique has been developed, it will be possible to follow the localized destruction of alveolar elastin that occurs during the development of emphysema.
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Materials and Methods Preparation of Dog Lung Elastin
Freshly excised lungs from normal beagles were lavaged and perfused with cold saline at 25 cm H2O pressure to remove alveolar macrophages, circulating and alveolar neutrophils which may be a source of elastin-degrading enzymes."3 The absence of these cells after lavage and perfusion was established by light-microscopic examination of samples taken by random stratified sampling of the lung. The blood vessels and alveolar spaces were observed to be free of neutrophils and mononuclear cells. The lungs were then dissected to remove the pleura, the major airways, and the major blood vessels. The dissected lung was then rapidly frozen in liquid nitrogen, broken into small pieces, and lyophilized. The dried lung was ground, and the fine powdery parenchymal tissue was then separated from any remaining pleura, major airways, and large blood vessels, since those components remained as large aggregates after grinding. The dry parenchymal tissue powder was added slowly to 4% aqueous sodium dodecyl sulfate (SDS) solution (60 g tissue per liter SDS) in a boiling water bath. The mixture was kept in the hot SDS for 10 min and stirred vigorously and then cooled slowly overnight with stirring. Most soluble lung parenchymal proteins were removed by this step. The residue was harvested by centrifugation at 8000 rpm for 15 min (10,500g) in a refrigerated Sorvall RC5B centrifuge set at 5 C, and the excess SDS was removed by washing the residue three times with cold water under the same centrifugation conditions. Elastin was purified from the residue by extraction with 0.1 N NaOH in a boiling water bath, as originally described by Lansing.14 The residue obtained from this step was washed to neutrality with glass distilled water, lyophilized and ground to a fine powder under liquid nitrogen. It was a fluffy, beige powder that was characterized as pure elastin by amino acid analysis, resistance to hydrolysis or solubilization by all tested proteases (trypsin, chymotrypsin, and collagenase) except pancreatic and neutrophil elastases. This elastin was used as described below and was also the source of pure elastin for the initial localization studies. Antibody Preparation and Characterization
Antiserums to elastin were prepared in sheep according to the procedure of Sykes and Chidlow.15 Antiserums were prepared against purified chick aortic elastin 16 and purified dog lung parenchymal elastin. Both antiserums were fractionated with ammonium sulfate (final concentration 33%) to prepare the IgG fraction and titered by the use of the radioimmunoassay of Christner et al."6 Using the antiserum prepared against chick aortic elastin and radioactive chick tropoelastin as the antigen, a 1 100 dilution of the antiserum precipitated 63.9% of the antigen in the radioimmunoassay. The antiserum against dog lung elastin precipitated only 30.0% of the antigen at the same antiserum dilution. These data suggested that antibodies raised against either antigen can bind to chick aortic tropoelastin, though the homologous species appeared to react more strongly. This was supported by absorption experiments that showed that chick aortic elastin bound its homologous antibody more completely than it bound the heterologous antidog lung elastin antibody. The reverse experiment also showed species selectivity in antibody binding. These preliminary findings are analyzed, using electron-microscopic approaches, in Results. Lung Tissue for Electron Microscopy
Excised lungs from normal beagles were lavaged and perfused with saline at 25 cm H2O pressure. They were fixed in inflation at 25 cm H20 at 4 C for a period of 4 hours with 2% glutaraldehyde in modified Millonig's phosphate buffer,17 pH 7.4, balanced by the addi-
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tion of NaCl or deionized water to 330 mOsm. The pleura, major airways, and major blood vessels were removed by dissection, and 1-cc blocks were cut from the remaining parenchyma. The blocks were placed in fresh 2% glutaraldehyde in Millonig's buffer (330 mOsm) overnight at 4 C. The blocks were then minced into 2-mm cubes, rinsed in Millonig's buffer three times, over a period of 30 minutes. They were then fixed in 1% OsO, in Sym-Collidine buffer, pH 7.4 (330 mOsm), for 1-4 hours.18 They were rinsed three times in Sym-Collidine buffer, dehydrated in graded ethanol solutions, cleared with propylene oxide, and embedded in Epon 812. Silver sections were cut with the Reichert Om-U2 ultramicrotome with a diamond knife. The effect of osmification time on the darkening of elastin was examined. In a modified procedure, excised dog lungs were lavaged and perfused with saline at 25 cm H20 pressure. They were fixed in inflation at 25 cm H20 at 4 C with 10% buffered commercial formalin for 4 hours. The pleura, major airways, and major blood vessels were removed by dissection and 1-cc blocks were cut from the remaining parenchyma. The blocks were placed in fresh 10% buffered formalin and fixed overnight in the cold. The blocks were minced into smaller blocks, and 2-mm cubes were rinsed in Millonig's buffer, dehydrated in graded ethanol solutions, cleared with propylene oxide, and embedded either in Epon 812 or Spurr's low viscosity medium. Silver sections were cut with the Reichert Om-U2 with a diamond knife. Several sections were taken, some of which were stained with uranyl acetate and lead citrate. Other sections were treated with silver TPPS, as described by Albert and Fleischer,3 to localize elastin. Immunologic Staining Preembedding Techniques
In preliminary experiments, major difficulties were encountered with nonspecific staining. However, it was found that pretreatment with a 5% bovine serum albumin (BSA) solution containing 0.1 M NaCl, 0.05 M sodium phosphate, and 0.01 M glycine,"9 pH 7.5, for 30 minutes reduced the background nonspecific ferritin staining and accentuated the specific staining. Isolated, lyophilized dog lung elastin particles were pretreated with the BSA solution. After BSA treatment, the control samples were treated with normal preimmune diluted sheep serum (1: 100) in 0.05 M Tris saline, pH 7.6, for 30 minutes at 4 C. The test samples were similarly treated with the sheep antielastins diluted 1:20 and 1: 100 in Tris saline. The antichick aortic elastin and the antidog lung elastin IgG were compared. All samples were then rinsed two times with Tris saline, treated again with BSA, and then treated directly with ferritin-conjugated rabbit antisheep IgG (Cappel Laboratories, Inc.) diluted 1: 100 for 30 minutes at 4 C. All samples were rinsed three times in Tris saline, fixed in 2% glutaraldehyde in Millonig's buffer, pH 7.3, dehydrated in graded concentrations of ethanol or 2,2-dimethoxypropane (DMP),20 and embedded in Spurr's low-viscosity embedding medium. All blocks were sectioned and examined directly with no further staining. Postembedding Technique
Isolated, lyophilized dog elastin was dehydrated in graded concentrations of ethanol or DMP and embedded in Spurr's low-viscosity medium. Silver sections were cut and picked up on either bare nickel or copper grids. They were then etched with water containing 0.01% benzene, 5 % methanol, and 5 % ethanol 12 for 5 minutes and then in water for 30 minutes at room temperature. The sections were then pretreated with BSA as described previously. They were then immersed in sequence in normal sheep IgG (control specimens) or sheep antielastin IgG. Antichick aorta and antidog lung elastin IgG were compared at 1 :20 and 1:100 dilution in Tris saline (4 C for 30 minutes). The antiserum treatment was followed by immersion in BSA solution (4 C for 5-10 minutes) and ferritin-conjugated rabbit antisheep IgG diluted 1:100 in Tris saline (4 C for 30 minutes). The sections were washed in
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water several times and then dried and examined directly without further staining. Several other sections were treated similarly except that the preliminary etching was eliminated to assess the effectiveness of pre-etching to enhance the final staining. Silver sections of normal dog lung obtained from blocks described in Materials and Methods were also pre-etched and then immunologically stained as described above. The lung blocks received either 1-hour or 4-hour postfixation in 1 % osmium to contrast the effect of ferritin on either light- or dark-appearing elastin. These sections were stained with uranyl acetate and lead citrate to develop contrast in other tissue components and then examined.
Results Standard Stains
The electron density of lung elastin in standard electron-microscopic procedures utilizing osmium as the secondary fixative is demonstrated in the sections of the dog lung shown in Figure 1. Amorphous elastin darkens with an extended 4-hour treatment in a 1% osmium tetroxide solution buffered with 0. 1 M Sym-Collidine solution, pH 7.4, as seen in Figure 1A. Shortening of the time to 1 hour or less in the osmium fixative results in a light staining appearance of elastin as seen in the reinforcing ring of the alveolar duct in Figure 1B. Elimination of the secondary fixation with OS04 results in the appearance of extremely electron-transparent elastin as seen at E in Figure 1C. The staining reaction of a serial section with silver TPPS is shown in Figure 1D. The elastin is stained black and is distinguishable from other connective tissue of the alveolar interstitium because of its general morphology. Collagen fibrils are also dark staining, but they are distinguished from elastin because of the highly organized structure of the collagen and characteristic banding. Preembedding Staining
Isolated dog lung elastin fragments treated initially with normal preimmune sheep serum and followed by ferritin-conjugated rabbit antisheep IgG prior to embedding without using BSA treatment showed nonspecific binding of ferritin to the peripheral regions of the elastin. Elimination of the nonspecific binding was achieved, as shown in Figure 2A, by pretreatment of the elastin with 5% BSA solution prior to applying the primary and the secondary antiserums in the procedure. Specific binding of ferritin after treatment with sheep antidog elastin IgG 1 : 100 (absorbance at 280 nm = 0.04) was observed at the peripheral regions of elastin, as shown in Figure 2B. At the same dilution of 1 : 100 (absorbance at 280 nm = 1.7) sheep antichick elastin IgG, when applied to isolated dog elastin, exhibited very little specific binding of ferritin (Figure 2C) in spite of the higher protein content in the antichick antiserum.
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Postembedding Staining
Thin sections of isolated dog lung elastin embedded in Spurr's medium showed some nonspecific binding of ferritin to both the elastin and the Spurr's medium in the postembedding procedure described, even though pretreatment with BSA was included (Figure 3A). Elastin appears more electron-transparent than Spurr's medium. Sections treated with antichick aortic elastin serum instead of preimmune serum demonstrated some specific binding of ferritin to elastin, as seen in Figure 3B. The merits of pre-etching the thin sections in the postembedded immunologic staining procedures were discussed by Sternberger.12 In the present work, preetched sections appeared to result in a more uniform distribution of ferritin binding to elastin, although it was not a necessary step to demonstrate specific binding of the antibodies to elastin. In a separate set of experiments, control samples of postembedded lung sections treated with a 1:20 dilution of normal sheep IgG showed a small amount of nonspecific binding of ferritin to the tissue section. Samples treated with the sheep antichick elastin IgG diluted 1:20 showed some specific binding of ferritin to elastin, although a considerable amount of nonspecific binding was also noted to other tissue components and to the embedding medium itself. The results showed that the chick aortic elastin antibodies did not bind well to the dog lung elastin, suggesting that there may be species or organ specificity for different elastins. Lack of affinity of chick aorta elastin antibodies for dog lung elastin was verified in experiments where the primary antiserum was diluted to 1 : 100. At this dilution, antidog lung elastin antibody gave strong specific binding (Figure 4B) and very little nonspecific binding in either the control samples or those treated with the antiserums (Figures 4A and 4B), while the antichick antibody gave no binding. Use of the sheep antidog lung elastin IgG at 1 : 100 dilution to localize elastin in normal dog lung sections is demonstrated in Figures 5A and 5B. The elastin in these sections developed a gray contrast as a result of the 1hour osmification treatment. Immunologic binding of ferritin to the elastin was readily apparent, as seen in Figure 5B. Little or no nonspecific binding is apparent in Figure 5A. The antidog elastin antibody did not react with other tissue components, such as the microfibrillar components marked M in Figure 6, collagen, marked C, or proteoglycan, normally present in the spaces between collagen and elastin. These observations support the suggestions made above that there is selective binding of the antidog lung elastin antibody to amorphous elastin in the alveolar interstitium. Extended osmification to 4 hours produced the dark-staining elastin as
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seen in Figure 7. Immunologic localization of ferritin was also clearly visible for this specimen. The results demonstrated that both glutaraldehyde and osmium could be used in the fixation procedure to optimize tissue preservation for electron microscopy without interference with the immunologic procedure. The choice of the embedding medium to be used (Epon 812 or Spurr's) or the method of dehydration had little effect on the final results. Discussion
The localization and defining of elastin in the lung with the electron microscope is of particular interest, since the etiology of pulmonary emphysema has focused on the destruction of elastin in the alveolar walls.13 Early changes visualized at the ultrastructural level with the electron microscope involve the gradual disruption of the organization of the interstitial components.12 Elastin fragments, once removed from the alveolar wall, lose their identity; and contrast alone is generally insufficient for positive identification. The experiments in this report demonstrate that specific antibody against insoluble dog lung amorphous elastin can be used in a staining procedure to localize dog lung elastin either by preembedding or postembedding procedures for electron microscopy. Antibodies against dog lung elastin bind specifically to reactive sites on the surface of isolated dog lung elastin fragments or on the surface of embedded, sectioned elastin either isolated or in the interstitium of the alveolar walls. The need for such a specific staining procedure for elastin was apparent, since elastin in lung is not well defined morphologically and standard staining methods which rely entirely on the development of electron density are not sufficiently specific to differentiate elastin from other electron-dense regions. The silver TPPS stain used in Figure 1D, for example, in addition to darkening elastin, has darkened microfibrils, collagen, and other tissue components. Immunologic localization with ferritin-conjugated rabbit antisheep IgG antiserum, which binds specifically to the antielastin antibodies already attached to elastin, is enhanced because of the ferritin's recognizable morphology as well as its electron density. Since the ferritin is an electron-dense marker, it was thought necessary to prevent darkening of the elastin usually encountered by osmification. Light-staining elastin was achieved by either eliminating osmification or by reducing the time of osmification to less than 1 hour. The present results, however, demonstrated that ferritin localization could be distinguished even in dark-staining elastin produced by extending the osmification times to 4 hours.
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A major problem encountered in all immunologic procedures is associated with the nonspecific binding of antiserums. Use of a protein such as BSA for pretreatment helped but did not completely insure the elimination of nonspecific binding of the primary antiserums to the embedding media or other tissue components. Dilution of the antiserums combined with the use of BSA, however, was effective in reducing nonspecific binding to a very low level. The highly specific sheep antidog lung elastin antibody made it possible in the homologous system to dilute the serums so that the background binding was virtually nonexistent. Thus, complete elimination of the nonspecific binding in postembedded sections offers the possibility of localizing small fragments of elastin in the alveolar spaces or within cells, eliminating the ambiguity associated with the presently used stains. Preembedding techniques have limited value in localizing elastin in the interstitial space of the whole lung, since penetration of either the primary antiserums or the ferritin-conjugated antibodies into the tissue and, in particular, into elastin is highly unlikely. In diseased or emphysematous lung in which elastin has become exposed to the alveolar space or otherwise detached from the alveolar septum, the antibodies can bind to the exposed surfaces. The appearance of ferritin binding to elastin in these cases, in addition to identifying exposed elastin, serves to identify gaps where elastin has been loosened from the interstitium or broken into fragments. Postembedding methods offer a decided advantage over preembedding methods, because they offer the possibility of serial sectioning to compare identical regions of the lung by different staining methods. The data presented in this paper establish the feasibility of using an immunologic approach for the identification of the amorphous component of canine alveolar interstitial elastin. This technique can now be expanded to identify and localize the microfibrillar component of elastin and other structural proteins within the lung. References 1. Lillie RD: Connective tissue fibers and membranes, Histopathologic Technique and Practical Histochemistry. Third edition. New York, Blakiston, 1965, p 551 2. Pease DC, Molinari S: Electron microscopy of muscular arteries: Pial vessels of the cat and monkey. J Ultrastruct Res 3:447-468, 1960 3. Albert EN, Fleischer E: A new electron-dense stain for elastic tissue. J Histochem Cytochem 18:697-708, 1970 4. Mizuhira V, Nakamura H, Fujioka T: New staining method for the elastic fibers using a tannic acid-glutaraldehyde mixture. J Electron Microsc (Tokyo) 21:240, 1972 5. Futaesaku Y, Mizuhira V, Nakamura H: A new method using tannic acid as an elastic fiber stain. Proc 4th Int Cong on Histochem and Cytochem, Kyoto, Japan, 1972, p 155
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6. Mizuhira V, Futaesaku Y, Nishi A: The new fixation method using tannic acid and ultrastructure of microtubules. J Electron Microsc (Tokyo) 21:240, 1972 7. Kajikawa K, Yamaguchi T, Katsuda S, Miwa A: An improved electron stain for elastic fibers using tannic acid. J Electron Microsc (Tokyo) 24:287-289, 1975 8. Singer SJ: Preparation of an electron-dense antibody conjugate. Nature 183:15231524, 1959 9. Singer SJ, Schick AF: The purpose of specific stains for electron microscopy prepared by the conjugation of antibody molecules with ferritin. J Biophys Biochem Cytol 9:519-537, 1961 10. Singer SJ, McLean JD: Ferritin-antibody conjugates: Stains for electron microscopy. Lab Invest 12:1002-1008, 1963 11. Painter RG, Takuyasu KT, Singer SJ: Immunoferritin localization of intracellular antigens: The use of ultracryotomy to obtain ultrathin sections suitable for direct immunoferritin staining. Proc Natl Acad Sci 70:1649-1653, 1973 12. Sternberger LA: The unlabelled-antibody-peroxidase and the quantitative immunuranium methods in light and electron immunohistochemistry, Techniques of Biochemical and Biophysical Morphology. Vol 1. New York, Wiley Intersciences, John Wiley & Sons, 1972, pp 67-83 13. Janoff A, Sloan B, Weinbaum G, Damiano V, Sandhaus RA, Elias J, Kimbel P: Experimental emphysema induced with purified human neutrophil elastase: Tissue localization of the instilled protease. Am Rev Resp Dis 115:461-478, 1977 14. Lansing Al, Rosenthal TB, Alex M, Dempsey EW: The structure and chemical characterization of elastic fibers as revealed by elastase and by electron microscopy. Anat Rec 114:555-575, 1952 15. Sykes BC, Chidlow JW: Precipitating antibodies directed against soluble elastin: The basis of a sensitive assay. FEBS Lett 47:222-224, 1974 16. Christner P, Dixon M, Cywinski A, Rosenbloom J: Radioimmunological identification of tropoelastin. Biochem J 157:525-528, 1976 17. Millonig G: Advantages of a phosphate buffer for OsO, solutions in fixation. J Appl Physics, 32:1637, 1961 18. Gil J, Weibel ER: The role of buffers in lung fixation with glutaraldehyde and osmium tetroxide. J Ultrastruct Res 25:331-348, 1968 19. McLean JD, Singer SJ: A technique for the specific staining of macromolecules and viruses with ferritin-antibody conjugates. J Mol Biol 56:633-635, 1971 20. Muller LL, Jacks TJ: Rapid chemical dehydration of samples for electron microscopic examinations. J Histochem Cytochem 23:107-110, 1975 21. Kuhn C, Yu S, Chraplyvy M, Linder HE, Senior RM: The induction of emphysema with elastase. Lab Invest 34:372-380, 1976
Acknowledgments The authors gratefully acknowledge the assistance afforded by Mr. Alan Sandler of the Franklin Research Center and Dr. Umberto Kucich and Miss Denise Damato of the Albert Einstein Medical Center.
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Figure 3A-lsolated dog lung elastin (E), postembedding, stained. Grids etched, BSAtreated and normal sheep serum and ferritin-conjugated rabbit antisheep IgG. Nonspecific binding of ferritin at arrows. (X95,000) B-Isolated dog elastin (E), postembedding, stained. Grids etched, BSA-treated and sheep antichick aorta elastin and ferritin-conjugated rabbit antisheep IgG. Specific binding of ferritin at arrows. (X95,000) (With a photographic reduction of 9%)
Figure 4A-Isolated dog lung elastin (E), postembedding, stained. Grids etched, BSAtreated and normal sheep serum 1:100 and ferritin-conjugated rabbit antisheep IgG. Nonspecific binding at arrows. (X51,000) B-Isolated dog lung elastin (E), postembedding, stained. Grids etched, BSA-treated. Sheep antidog lung elastin 1:100 and ferritin-conjugated rabbit antisheep IgG. Specific binding to elastin. (X51,000) (With a photographic reduction of 10%)
Figure 5A-Normal dog lung, postembedding, stained. Grids etched, BSA-treated and normal sheep serum 1:100 and ferritin-conjugated rabbit antisheep IgG. Final stain B-Normal dog lung, posturanyl acetate and lead citrate. Elastin (E). (X51,000) embedding, stained. Grids etched, BSA-treated and sheep antidog lung elastin 1:100 and ferritin conjugated rabbit antisheep IgG. Final stain uranyl acetate and lead citrate. Specific binding of ferritin to elastin (E). (X51,000) (With a photographic reduction of 4%)
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[End of Article]
Research on the pathogenesis of experimental emphysema has involved studies of the distribution of and destruction of elastin in the alveolar interstitium. The ill-defined organization of elastin in the alveolar interstitium makes it difficult to identify the elastin specifically by staining procedures ordinarily used for electron microscopy. This problem becomes more significant when the elastic tissue is fragmented during emphysema development and localization of the elastin fragments is essential. Therefore, a specific technique using high-titer antibodies against purified canine lung elastin was developed. The primary antibody was used on preembedded or etched postembedded sections. Localization of the antielastin IgG was accomplished with ferritin-labeled rabbit antisheep IgG as the secondary antibody. Treatment with the preimmune serum gave negligible ferritin background staining. The antielastin antibody did not react with lung connective tissue proteins such as the microfibrillar component of elastin or collagen or proteoglycan. The antielastin antibody appeared to be species specific. The method may be useful for studies of experimental emphysema. (Am J Pathol 96:439456, 1979)
RECENT EMPHASIS in research on the etiology and pathogenesis of experimental pulmonary emphysema has been directed toward electron-microscopic studies of the distribution and destruction of elastin in the interstitial spaces of the alveolar walls. In order to accomplish such an analysis, specific electron-dense stains to visualize elastin in lung at the ultrastructural level have become increasingly important. With the use of standard electron-microscopic procedures involving primary fixation with glutaraldehyde and secondary fixation with osmium tetroxide, elastin is variably darkened, primarily because of the osmiophilic nature of elastin.1 Some control of the darkening effect may be achieved by control of the osmification time, but variability in the contrast ultimately depends upon the rate of penetration of osmium into the tissue. Early attempts to localize elastin by electron microscopy involved the use of phosphotungstic acid (PTA) (0. 1% in 50% ethanol).2 PTA combines vigorously with both elastin and collagen, but elastin is distinguished by the absence of a periodic structure characteristic of collagen. Since PTA From the Franklin Research Center, Philadelphia, Pennsylvania; the University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania; and the Albert Einstein Medical Center, Pulmonary Disease Section, Philadelphia, Pennsylvania. Supported by grants from the Council for Tobacco Research, Inc.-USA (Grant 901A) and the National Institutes of Health (Grants AM-02863, AM-02553, DE-02623, and PO1-HL-20994). Accepted for publication Marci 14, 1979. Address reprint requests to V. V. Damiano, PhD, Principal Scientist, Physics and Material Section, Franklin Research Center, Philadelphia, PA 19103. 0002-9440/79/0809-0439$01.00 439 O American Association of Pathologists
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extracts the reduced osmium from the thin sections, effective contrast, which is developed for other cellular components, is generally lost. Consequently, PTA is not effective in localizing elastin. Albert and Fleischer 3 developed a stain somewhat more specific for elastin by using silver salts of the sulfonated tetraphenol porphine (TPPS). They reported effective localization of elastin in both newborn and adult mouse aortas. More recently, a stain has been suggested for elastin, utilizing tannic acid-glutaraldehyde mixtures as the primary fixative. This was developed for the aorta, small arteries, cartilage, and bone connective tissue.4'5'6 A modification of this technique was used 7 by applying the tannic acid with uranyl acetate as a stain to postembedded (Epon 812) thin sections of chick aorta. All of the methods developed to date to localize elastin in tissue rely upon the imparting of an electron-dense stain of variable intensity and distribution to the elastin. Positive identification is based in part upon the relative association of the elastin with other components in the interstitial space and in part upon the contrast developed by the specific stain. Isolated elastin fragments encountered in emphysematous lung are difficult to identify positively on the basis of electron-dense contrast alone, since other osmiophilic components of the tissue, such as fibrin, chromatin, or lipids may develop similar electron-dense contrast and be mistaken for fragmented elastin. Immunologic staining procedures that utilize a well-defined marker such as ferritin offer the best potential method for locating specific cellular or tissue components,8'9"10 since the marker morphology and unique specificity as well as its electron density serve to specifically identify the component with which the antibody reacts. Immunologic stains, when applied directly to thin postembedded sections,"1"12 obviate the requirement of antibody penetration through cellular membranes or the dense interstitial space and offer the best approach to localization. Postembedding staining applied to serial sections further offers the possibility of applying different immunologic stains to sequential sections to localize more than one component in the same area. The objective of the present investigation was to develop an immunologic staining procedure that would specifically localize isolated elastin or elastin in the alveolar wall of postembedded thin sections prepared for electron microscopy. Once such a technique has been developed, it will be possible to follow the localized destruction of alveolar elastin that occurs during the development of emphysema.
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Materials and Methods Preparation of Dog Lung Elastin
Freshly excised lungs from normal beagles were lavaged and perfused with cold saline at 25 cm H2O pressure to remove alveolar macrophages, circulating and alveolar neutrophils which may be a source of elastin-degrading enzymes."3 The absence of these cells after lavage and perfusion was established by light-microscopic examination of samples taken by random stratified sampling of the lung. The blood vessels and alveolar spaces were observed to be free of neutrophils and mononuclear cells. The lungs were then dissected to remove the pleura, the major airways, and the major blood vessels. The dissected lung was then rapidly frozen in liquid nitrogen, broken into small pieces, and lyophilized. The dried lung was ground, and the fine powdery parenchymal tissue was then separated from any remaining pleura, major airways, and large blood vessels, since those components remained as large aggregates after grinding. The dry parenchymal tissue powder was added slowly to 4% aqueous sodium dodecyl sulfate (SDS) solution (60 g tissue per liter SDS) in a boiling water bath. The mixture was kept in the hot SDS for 10 min and stirred vigorously and then cooled slowly overnight with stirring. Most soluble lung parenchymal proteins were removed by this step. The residue was harvested by centrifugation at 8000 rpm for 15 min (10,500g) in a refrigerated Sorvall RC5B centrifuge set at 5 C, and the excess SDS was removed by washing the residue three times with cold water under the same centrifugation conditions. Elastin was purified from the residue by extraction with 0.1 N NaOH in a boiling water bath, as originally described by Lansing.14 The residue obtained from this step was washed to neutrality with glass distilled water, lyophilized and ground to a fine powder under liquid nitrogen. It was a fluffy, beige powder that was characterized as pure elastin by amino acid analysis, resistance to hydrolysis or solubilization by all tested proteases (trypsin, chymotrypsin, and collagenase) except pancreatic and neutrophil elastases. This elastin was used as described below and was also the source of pure elastin for the initial localization studies. Antibody Preparation and Characterization
Antiserums to elastin were prepared in sheep according to the procedure of Sykes and Chidlow.15 Antiserums were prepared against purified chick aortic elastin 16 and purified dog lung parenchymal elastin. Both antiserums were fractionated with ammonium sulfate (final concentration 33%) to prepare the IgG fraction and titered by the use of the radioimmunoassay of Christner et al."6 Using the antiserum prepared against chick aortic elastin and radioactive chick tropoelastin as the antigen, a 1 100 dilution of the antiserum precipitated 63.9% of the antigen in the radioimmunoassay. The antiserum against dog lung elastin precipitated only 30.0% of the antigen at the same antiserum dilution. These data suggested that antibodies raised against either antigen can bind to chick aortic tropoelastin, though the homologous species appeared to react more strongly. This was supported by absorption experiments that showed that chick aortic elastin bound its homologous antibody more completely than it bound the heterologous antidog lung elastin antibody. The reverse experiment also showed species selectivity in antibody binding. These preliminary findings are analyzed, using electron-microscopic approaches, in Results. Lung Tissue for Electron Microscopy
Excised lungs from normal beagles were lavaged and perfused with saline at 25 cm H2O pressure. They were fixed in inflation at 25 cm H20 at 4 C for a period of 4 hours with 2% glutaraldehyde in modified Millonig's phosphate buffer,17 pH 7.4, balanced by the addi-
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tion of NaCl or deionized water to 330 mOsm. The pleura, major airways, and major blood vessels were removed by dissection, and 1-cc blocks were cut from the remaining parenchyma. The blocks were placed in fresh 2% glutaraldehyde in Millonig's buffer (330 mOsm) overnight at 4 C. The blocks were then minced into 2-mm cubes, rinsed in Millonig's buffer three times, over a period of 30 minutes. They were then fixed in 1% OsO, in Sym-Collidine buffer, pH 7.4 (330 mOsm), for 1-4 hours.18 They were rinsed three times in Sym-Collidine buffer, dehydrated in graded ethanol solutions, cleared with propylene oxide, and embedded in Epon 812. Silver sections were cut with the Reichert Om-U2 ultramicrotome with a diamond knife. The effect of osmification time on the darkening of elastin was examined. In a modified procedure, excised dog lungs were lavaged and perfused with saline at 25 cm H20 pressure. They were fixed in inflation at 25 cm H20 at 4 C with 10% buffered commercial formalin for 4 hours. The pleura, major airways, and major blood vessels were removed by dissection and 1-cc blocks were cut from the remaining parenchyma. The blocks were placed in fresh 10% buffered formalin and fixed overnight in the cold. The blocks were minced into smaller blocks, and 2-mm cubes were rinsed in Millonig's buffer, dehydrated in graded ethanol solutions, cleared with propylene oxide, and embedded either in Epon 812 or Spurr's low viscosity medium. Silver sections were cut with the Reichert Om-U2 with a diamond knife. Several sections were taken, some of which were stained with uranyl acetate and lead citrate. Other sections were treated with silver TPPS, as described by Albert and Fleischer,3 to localize elastin. Immunologic Staining Preembedding Techniques
In preliminary experiments, major difficulties were encountered with nonspecific staining. However, it was found that pretreatment with a 5% bovine serum albumin (BSA) solution containing 0.1 M NaCl, 0.05 M sodium phosphate, and 0.01 M glycine,"9 pH 7.5, for 30 minutes reduced the background nonspecific ferritin staining and accentuated the specific staining. Isolated, lyophilized dog lung elastin particles were pretreated with the BSA solution. After BSA treatment, the control samples were treated with normal preimmune diluted sheep serum (1: 100) in 0.05 M Tris saline, pH 7.6, for 30 minutes at 4 C. The test samples were similarly treated with the sheep antielastins diluted 1:20 and 1: 100 in Tris saline. The antichick aortic elastin and the antidog lung elastin IgG were compared. All samples were then rinsed two times with Tris saline, treated again with BSA, and then treated directly with ferritin-conjugated rabbit antisheep IgG (Cappel Laboratories, Inc.) diluted 1: 100 for 30 minutes at 4 C. All samples were rinsed three times in Tris saline, fixed in 2% glutaraldehyde in Millonig's buffer, pH 7.3, dehydrated in graded concentrations of ethanol or 2,2-dimethoxypropane (DMP),20 and embedded in Spurr's low-viscosity embedding medium. All blocks were sectioned and examined directly with no further staining. Postembedding Technique
Isolated, lyophilized dog elastin was dehydrated in graded concentrations of ethanol or DMP and embedded in Spurr's low-viscosity medium. Silver sections were cut and picked up on either bare nickel or copper grids. They were then etched with water containing 0.01% benzene, 5 % methanol, and 5 % ethanol 12 for 5 minutes and then in water for 30 minutes at room temperature. The sections were then pretreated with BSA as described previously. They were then immersed in sequence in normal sheep IgG (control specimens) or sheep antielastin IgG. Antichick aorta and antidog lung elastin IgG were compared at 1 :20 and 1:100 dilution in Tris saline (4 C for 30 minutes). The antiserum treatment was followed by immersion in BSA solution (4 C for 5-10 minutes) and ferritin-conjugated rabbit antisheep IgG diluted 1:100 in Tris saline (4 C for 30 minutes). The sections were washed in
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water several times and then dried and examined directly without further staining. Several other sections were treated similarly except that the preliminary etching was eliminated to assess the effectiveness of pre-etching to enhance the final staining. Silver sections of normal dog lung obtained from blocks described in Materials and Methods were also pre-etched and then immunologically stained as described above. The lung blocks received either 1-hour or 4-hour postfixation in 1 % osmium to contrast the effect of ferritin on either light- or dark-appearing elastin. These sections were stained with uranyl acetate and lead citrate to develop contrast in other tissue components and then examined.
Results Standard Stains
The electron density of lung elastin in standard electron-microscopic procedures utilizing osmium as the secondary fixative is demonstrated in the sections of the dog lung shown in Figure 1. Amorphous elastin darkens with an extended 4-hour treatment in a 1% osmium tetroxide solution buffered with 0. 1 M Sym-Collidine solution, pH 7.4, as seen in Figure 1A. Shortening of the time to 1 hour or less in the osmium fixative results in a light staining appearance of elastin as seen in the reinforcing ring of the alveolar duct in Figure 1B. Elimination of the secondary fixation with OS04 results in the appearance of extremely electron-transparent elastin as seen at E in Figure 1C. The staining reaction of a serial section with silver TPPS is shown in Figure 1D. The elastin is stained black and is distinguishable from other connective tissue of the alveolar interstitium because of its general morphology. Collagen fibrils are also dark staining, but they are distinguished from elastin because of the highly organized structure of the collagen and characteristic banding. Preembedding Staining
Isolated dog lung elastin fragments treated initially with normal preimmune sheep serum and followed by ferritin-conjugated rabbit antisheep IgG prior to embedding without using BSA treatment showed nonspecific binding of ferritin to the peripheral regions of the elastin. Elimination of the nonspecific binding was achieved, as shown in Figure 2A, by pretreatment of the elastin with 5% BSA solution prior to applying the primary and the secondary antiserums in the procedure. Specific binding of ferritin after treatment with sheep antidog elastin IgG 1 : 100 (absorbance at 280 nm = 0.04) was observed at the peripheral regions of elastin, as shown in Figure 2B. At the same dilution of 1 : 100 (absorbance at 280 nm = 1.7) sheep antichick elastin IgG, when applied to isolated dog elastin, exhibited very little specific binding of ferritin (Figure 2C) in spite of the higher protein content in the antichick antiserum.
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Postembedding Staining
Thin sections of isolated dog lung elastin embedded in Spurr's medium showed some nonspecific binding of ferritin to both the elastin and the Spurr's medium in the postembedding procedure described, even though pretreatment with BSA was included (Figure 3A). Elastin appears more electron-transparent than Spurr's medium. Sections treated with antichick aortic elastin serum instead of preimmune serum demonstrated some specific binding of ferritin to elastin, as seen in Figure 3B. The merits of pre-etching the thin sections in the postembedded immunologic staining procedures were discussed by Sternberger.12 In the present work, preetched sections appeared to result in a more uniform distribution of ferritin binding to elastin, although it was not a necessary step to demonstrate specific binding of the antibodies to elastin. In a separate set of experiments, control samples of postembedded lung sections treated with a 1:20 dilution of normal sheep IgG showed a small amount of nonspecific binding of ferritin to the tissue section. Samples treated with the sheep antichick elastin IgG diluted 1:20 showed some specific binding of ferritin to elastin, although a considerable amount of nonspecific binding was also noted to other tissue components and to the embedding medium itself. The results showed that the chick aortic elastin antibodies did not bind well to the dog lung elastin, suggesting that there may be species or organ specificity for different elastins. Lack of affinity of chick aorta elastin antibodies for dog lung elastin was verified in experiments where the primary antiserum was diluted to 1 : 100. At this dilution, antidog lung elastin antibody gave strong specific binding (Figure 4B) and very little nonspecific binding in either the control samples or those treated with the antiserums (Figures 4A and 4B), while the antichick antibody gave no binding. Use of the sheep antidog lung elastin IgG at 1 : 100 dilution to localize elastin in normal dog lung sections is demonstrated in Figures 5A and 5B. The elastin in these sections developed a gray contrast as a result of the 1hour osmification treatment. Immunologic binding of ferritin to the elastin was readily apparent, as seen in Figure 5B. Little or no nonspecific binding is apparent in Figure 5A. The antidog elastin antibody did not react with other tissue components, such as the microfibrillar components marked M in Figure 6, collagen, marked C, or proteoglycan, normally present in the spaces between collagen and elastin. These observations support the suggestions made above that there is selective binding of the antidog lung elastin antibody to amorphous elastin in the alveolar interstitium. Extended osmification to 4 hours produced the dark-staining elastin as
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seen in Figure 7. Immunologic localization of ferritin was also clearly visible for this specimen. The results demonstrated that both glutaraldehyde and osmium could be used in the fixation procedure to optimize tissue preservation for electron microscopy without interference with the immunologic procedure. The choice of the embedding medium to be used (Epon 812 or Spurr's) or the method of dehydration had little effect on the final results. Discussion
The localization and defining of elastin in the lung with the electron microscope is of particular interest, since the etiology of pulmonary emphysema has focused on the destruction of elastin in the alveolar walls.13 Early changes visualized at the ultrastructural level with the electron microscope involve the gradual disruption of the organization of the interstitial components.12 Elastin fragments, once removed from the alveolar wall, lose their identity; and contrast alone is generally insufficient for positive identification. The experiments in this report demonstrate that specific antibody against insoluble dog lung amorphous elastin can be used in a staining procedure to localize dog lung elastin either by preembedding or postembedding procedures for electron microscopy. Antibodies against dog lung elastin bind specifically to reactive sites on the surface of isolated dog lung elastin fragments or on the surface of embedded, sectioned elastin either isolated or in the interstitium of the alveolar walls. The need for such a specific staining procedure for elastin was apparent, since elastin in lung is not well defined morphologically and standard staining methods which rely entirely on the development of electron density are not sufficiently specific to differentiate elastin from other electron-dense regions. The silver TPPS stain used in Figure 1D, for example, in addition to darkening elastin, has darkened microfibrils, collagen, and other tissue components. Immunologic localization with ferritin-conjugated rabbit antisheep IgG antiserum, which binds specifically to the antielastin antibodies already attached to elastin, is enhanced because of the ferritin's recognizable morphology as well as its electron density. Since the ferritin is an electron-dense marker, it was thought necessary to prevent darkening of the elastin usually encountered by osmification. Light-staining elastin was achieved by either eliminating osmification or by reducing the time of osmification to less than 1 hour. The present results, however, demonstrated that ferritin localization could be distinguished even in dark-staining elastin produced by extending the osmification times to 4 hours.
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A major problem encountered in all immunologic procedures is associated with the nonspecific binding of antiserums. Use of a protein such as BSA for pretreatment helped but did not completely insure the elimination of nonspecific binding of the primary antiserums to the embedding media or other tissue components. Dilution of the antiserums combined with the use of BSA, however, was effective in reducing nonspecific binding to a very low level. The highly specific sheep antidog lung elastin antibody made it possible in the homologous system to dilute the serums so that the background binding was virtually nonexistent. Thus, complete elimination of the nonspecific binding in postembedded sections offers the possibility of localizing small fragments of elastin in the alveolar spaces or within cells, eliminating the ambiguity associated with the presently used stains. Preembedding techniques have limited value in localizing elastin in the interstitial space of the whole lung, since penetration of either the primary antiserums or the ferritin-conjugated antibodies into the tissue and, in particular, into elastin is highly unlikely. In diseased or emphysematous lung in which elastin has become exposed to the alveolar space or otherwise detached from the alveolar septum, the antibodies can bind to the exposed surfaces. The appearance of ferritin binding to elastin in these cases, in addition to identifying exposed elastin, serves to identify gaps where elastin has been loosened from the interstitium or broken into fragments. Postembedding methods offer a decided advantage over preembedding methods, because they offer the possibility of serial sectioning to compare identical regions of the lung by different staining methods. The data presented in this paper establish the feasibility of using an immunologic approach for the identification of the amorphous component of canine alveolar interstitial elastin. This technique can now be expanded to identify and localize the microfibrillar component of elastin and other structural proteins within the lung. References 1. Lillie RD: Connective tissue fibers and membranes, Histopathologic Technique and Practical Histochemistry. Third edition. New York, Blakiston, 1965, p 551 2. Pease DC, Molinari S: Electron microscopy of muscular arteries: Pial vessels of the cat and monkey. J Ultrastruct Res 3:447-468, 1960 3. Albert EN, Fleischer E: A new electron-dense stain for elastic tissue. J Histochem Cytochem 18:697-708, 1970 4. Mizuhira V, Nakamura H, Fujioka T: New staining method for the elastic fibers using a tannic acid-glutaraldehyde mixture. J Electron Microsc (Tokyo) 21:240, 1972 5. Futaesaku Y, Mizuhira V, Nakamura H: A new method using tannic acid as an elastic fiber stain. Proc 4th Int Cong on Histochem and Cytochem, Kyoto, Japan, 1972, p 155
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6. Mizuhira V, Futaesaku Y, Nishi A: The new fixation method using tannic acid and ultrastructure of microtubules. J Electron Microsc (Tokyo) 21:240, 1972 7. Kajikawa K, Yamaguchi T, Katsuda S, Miwa A: An improved electron stain for elastic fibers using tannic acid. J Electron Microsc (Tokyo) 24:287-289, 1975 8. Singer SJ: Preparation of an electron-dense antibody conjugate. Nature 183:15231524, 1959 9. Singer SJ, Schick AF: The purpose of specific stains for electron microscopy prepared by the conjugation of antibody molecules with ferritin. J Biophys Biochem Cytol 9:519-537, 1961 10. Singer SJ, McLean JD: Ferritin-antibody conjugates: Stains for electron microscopy. Lab Invest 12:1002-1008, 1963 11. Painter RG, Takuyasu KT, Singer SJ: Immunoferritin localization of intracellular antigens: The use of ultracryotomy to obtain ultrathin sections suitable for direct immunoferritin staining. Proc Natl Acad Sci 70:1649-1653, 1973 12. Sternberger LA: The unlabelled-antibody-peroxidase and the quantitative immunuranium methods in light and electron immunohistochemistry, Techniques of Biochemical and Biophysical Morphology. Vol 1. New York, Wiley Intersciences, John Wiley & Sons, 1972, pp 67-83 13. Janoff A, Sloan B, Weinbaum G, Damiano V, Sandhaus RA, Elias J, Kimbel P: Experimental emphysema induced with purified human neutrophil elastase: Tissue localization of the instilled protease. Am Rev Resp Dis 115:461-478, 1977 14. Lansing Al, Rosenthal TB, Alex M, Dempsey EW: The structure and chemical characterization of elastic fibers as revealed by elastase and by electron microscopy. Anat Rec 114:555-575, 1952 15. Sykes BC, Chidlow JW: Precipitating antibodies directed against soluble elastin: The basis of a sensitive assay. FEBS Lett 47:222-224, 1974 16. Christner P, Dixon M, Cywinski A, Rosenbloom J: Radioimmunological identification of tropoelastin. Biochem J 157:525-528, 1976 17. Millonig G: Advantages of a phosphate buffer for OsO, solutions in fixation. J Appl Physics, 32:1637, 1961 18. Gil J, Weibel ER: The role of buffers in lung fixation with glutaraldehyde and osmium tetroxide. J Ultrastruct Res 25:331-348, 1968 19. McLean JD, Singer SJ: A technique for the specific staining of macromolecules and viruses with ferritin-antibody conjugates. J Mol Biol 56:633-635, 1971 20. Muller LL, Jacks TJ: Rapid chemical dehydration of samples for electron microscopic examinations. J Histochem Cytochem 23:107-110, 1975 21. Kuhn C, Yu S, Chraplyvy M, Linder HE, Senior RM: The induction of emphysema with elastase. Lab Invest 34:372-380, 1976
Acknowledgments The authors gratefully acknowledge the assistance afforded by Mr. Alan Sandler of the Franklin Research Center and Dr. Umberto Kucich and Miss Denise Damato of the Albert Einstein Medical Center.
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Figure 3A-lsolated dog lung elastin (E), postembedding, stained. Grids etched, BSAtreated and normal sheep serum and ferritin-conjugated rabbit antisheep IgG. Nonspecific binding of ferritin at arrows. (X95,000) B-Isolated dog elastin (E), postembedding, stained. Grids etched, BSA-treated and sheep antichick aorta elastin and ferritin-conjugated rabbit antisheep IgG. Specific binding of ferritin at arrows. (X95,000) (With a photographic reduction of 9%)
Figure 4A-Isolated dog lung elastin (E), postembedding, stained. Grids etched, BSAtreated and normal sheep serum 1:100 and ferritin-conjugated rabbit antisheep IgG. Nonspecific binding at arrows. (X51,000) B-Isolated dog lung elastin (E), postembedding, stained. Grids etched, BSA-treated. Sheep antidog lung elastin 1:100 and ferritin-conjugated rabbit antisheep IgG. Specific binding to elastin. (X51,000) (With a photographic reduction of 10%)
Figure 5A-Normal dog lung, postembedding, stained. Grids etched, BSA-treated and normal sheep serum 1:100 and ferritin-conjugated rabbit antisheep IgG. Final stain B-Normal dog lung, posturanyl acetate and lead citrate. Elastin (E). (X51,000) embedding, stained. Grids etched, BSA-treated and sheep antidog lung elastin 1:100 and ferritin conjugated rabbit antisheep IgG. Final stain uranyl acetate and lead citrate. Specific binding of ferritin to elastin (E). (X51,000) (With a photographic reduction of 4%)
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[End of Article]
