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glutaraldehyde fixation
TISSUE & CELL 19757 (I) 211-216 Published by Longman Group Ltd. Printed in Great Britain
M. LOCKE and P. HUIE
STAINING OF THE ELASTIC FIBERS IN INSECT CONNECTIVE TISSUE AFTER TANNIC ACID/GLUTARALDEHYDE FIXATION ABSTRACT. Locust neural lamella and Calpodes connect ive tissue fixed in glutaraldehyde have a fibrous component which stains after reaction with DAB and osmication and after stain ing sections with PTA. The fibers also stain when fixed in glutaraldehyde with tannic acid followed by osmication and section staining with lead citrate.
A COMPONENT of the acellular connective tissue from Calpodes ethlius becomes osmiophilic when tissue is reacted with Diaminobenzidine prior to osmication (Locke and Huie, 1972). This component also reacts with phosphotungstic acid when sections of unosmicated glutaraldehyde fixed tissue are stained with 2 % aqueous phosphotungstic acid. The occurrence of the fibers in connective tissue around the heart and nerve cord and their structural similarity to vertebrate elastic tissue where the fibers also stain with PTA (Greenlee et al., 1966; Ross and Bornstein, 1969), suggested that the stained components from Calpodes were elastic fibers. This paper reports another staining procedure for demonstrating these fibers and their occurrence in the neural lamella of female locusts. The connective tissue sheath around the nerve cord of cockroaches and silkworms is known to be elastic, and strongly birefringent in a way not accountable for by the collagen component (Richards and Schneider, 1958). Female locusts stretch their abdomens from 2'5 to 8-9 ems in order to oviposit, with considerable stretching of the nerve cord (Vincent and Wood , 1972; Vincent and Prentice, 1973). For these reasons we exDepartment of Zoology, Uni versity of Western Ontario, London, Canada. Received 26 August 1974.
pected to find elastic fibers in the locust neural lamella. When preliminary tests showed the fibers to be present, locust (Locusta migratoria) nerve cords were used as test material as well as the heart and nerve cord of Calpodes. Tissue was fixed in either 5 % glutaraldehyde buffered at pH 7·3 with 0·05 M cacodylate or in 2'5 % glutaraldehyde + 5 % tannic acid neutralized with NaOH and buffered with 0·05 M sodium phosphate to pH 7·2 (Tilney et al., 1973). Further treatments are described in the figure legends. All the plates have had comparable photographic processing. The female locust nerve cord has a well developed ring of axially oriented bundles of elastic fibers embedded in matrix just below the surface and outside the layer of collagen fibers in the neural lamella. These show up very clearly after incubating glutaraldehyde fixed tissue in DAB without H202 (Fig. I). The reaction is so intense that even when the sections are stained in uranyl acetate and lead citrate to bring up the background, the fibers still stand out (Figs. 2, 3). PTA on sections of glutaraldehyde fixed tissue stains the periphery of the fibers intensely (F ig. 4). Uranyl acetate and lead citrate have no such specificity and barely allow the fibers to be resolved (Fig. 5). Glutaraldehyde fixed tissue which has been post osmicated and then incubated in 2 % uranyl acetate overnight at room 211
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temperature (Locke et al., 1971) stains the fibers more than other connective tissue components (Fig. 6) but is less selective. Fig. 7 shows fibers from a connective tissue strand in Calpodes which has been fixed in tannic acid/glutaraldehyde, osmicated and the sections stained with lead citrate. Fibers are obvious in connective tissue from various sources after this treatment such as that below the epidermis, where they were previously unsuspected. From these results it seems probable that elastic fibers may be widespread in insect connective tissue. The staining procedures described do not all show up the same components. The PTA on sections and the uranyl acetate on osmicated tissue react with the surface of the large fibrous component giving density around less
dense fibers. The PTA in particular reacts to give dense amorphous material. The DAB and the tannic acid on the other hand cause the heavy metals (lead or osmium) to react with the whole fiber which appears uniformly dense and rodlike. Presumably the tannic acid cross links a variety of proteins in competition with glutaraldehyde and in particular those of the elastic fibers. These proteins then have free aromatic hydroxyl groups which react with heavy metals. Acknowledgements This work was generously supported by the National Research Council of Canada, Grant no. A6607.
m-matrix c-s-collagen f---elastic fibers h-haemocoel gl-glial cell Figs. 1-6. Adult female locust neural lamella transverse sections. Fig. 7, Calpodes pericardial connective tissue fiber. Fig. I. Glutaraldehyde fixation, DAB reaction without H 2 0 2 , osmication. x 11,600. Figs. 2, 3. As Fig. 1 but section stained with uranyl acetate and lead citrate. Fig. 2, x 11,000. Fig. 3, x 39,000. Fig. 4. Glutaraldehyde fixation, section stained with aqueous PTA. x 39,000. Fig. 5. Glutaraldehyde fixation, section stained with uranyl acetate and lead citrate. x 39,000. Fig. 6. Glutaraldehyde fixation, osmication, tissue stained with uranyl acetate. x 41,000. Fig. 7. Glutaraldehyde and tannic acid fixation, section stained with lead citrate. x 82,000.
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References GREENLEE, T. K., Ross, R. and HARTMAN, J. L. 1966. The fine structure of elastic fibers. J. Cell BioI., 30 (I), 59-71. LOCKE, M., KRISHNAN, N. and McMAHON, J. 1971. A routine method of obtaining high contrast without staining sections. J. Cell BioI., 50, 540-544. LOCKE, M. and HUIE, P. 1972. The fiber components of insect connective tissue. Tissue & Cell, 4 (4), 601-612. RICHARDS, V. A. G. and SCHNEIDER, D. 1958. Uber den komplexen Bau der Membranen des Bindegewebes von Insekten. Z. Naturf., 13 (10), 680-687. Ross, R. and BORNSTEIN, P. 1969. The elastic fiber. 1. The separation and partial characterization of its macromolecular components. J. Cell BioI., 40 (2), 366-381. TILNEY, L. G., BRYAN, J., BUSH, D. J., FUJIWARA, K., MOOSEKER, M. S., MURPHY, D. B. and SNYDER, D. H. (1973). Microtubules: Evidence for 13 protofilaments. J. Cell BioI., 59, 267-275. VINCENT, J. F. and WOOD, S. D. (1972). Mechanisms of abdominal extension during oviposition in Locus/a. Nature, Lond., 235 (5334), 167-168. VINCENT, J. F. V. and PRENTICE, J. H. (1973). Rheological properties of the extensible intersegmental membrane of the adult female locust. J. Mat, Sci., 8, 624-630.
M. LOCKE and P. HUIE
STAINING OF THE ELASTIC FIBERS IN INSECT CONNECTIVE TISSUE AFTER TANNIC ACID/GLUTARALDEHYDE FIXATION ABSTRACT. Locust neural lamella and Calpodes connect ive tissue fixed in glutaraldehyde have a fibrous component which stains after reaction with DAB and osmication and after stain ing sections with PTA. The fibers also stain when fixed in glutaraldehyde with tannic acid followed by osmication and section staining with lead citrate.
A COMPONENT of the acellular connective tissue from Calpodes ethlius becomes osmiophilic when tissue is reacted with Diaminobenzidine prior to osmication (Locke and Huie, 1972). This component also reacts with phosphotungstic acid when sections of unosmicated glutaraldehyde fixed tissue are stained with 2 % aqueous phosphotungstic acid. The occurrence of the fibers in connective tissue around the heart and nerve cord and their structural similarity to vertebrate elastic tissue where the fibers also stain with PTA (Greenlee et al., 1966; Ross and Bornstein, 1969), suggested that the stained components from Calpodes were elastic fibers. This paper reports another staining procedure for demonstrating these fibers and their occurrence in the neural lamella of female locusts. The connective tissue sheath around the nerve cord of cockroaches and silkworms is known to be elastic, and strongly birefringent in a way not accountable for by the collagen component (Richards and Schneider, 1958). Female locusts stretch their abdomens from 2'5 to 8-9 ems in order to oviposit, with considerable stretching of the nerve cord (Vincent and Wood , 1972; Vincent and Prentice, 1973). For these reasons we exDepartment of Zoology, Uni versity of Western Ontario, London, Canada. Received 26 August 1974.
pected to find elastic fibers in the locust neural lamella. When preliminary tests showed the fibers to be present, locust (Locusta migratoria) nerve cords were used as test material as well as the heart and nerve cord of Calpodes. Tissue was fixed in either 5 % glutaraldehyde buffered at pH 7·3 with 0·05 M cacodylate or in 2'5 % glutaraldehyde + 5 % tannic acid neutralized with NaOH and buffered with 0·05 M sodium phosphate to pH 7·2 (Tilney et al., 1973). Further treatments are described in the figure legends. All the plates have had comparable photographic processing. The female locust nerve cord has a well developed ring of axially oriented bundles of elastic fibers embedded in matrix just below the surface and outside the layer of collagen fibers in the neural lamella. These show up very clearly after incubating glutaraldehyde fixed tissue in DAB without H202 (Fig. I). The reaction is so intense that even when the sections are stained in uranyl acetate and lead citrate to bring up the background, the fibers still stand out (Figs. 2, 3). PTA on sections of glutaraldehyde fixed tissue stains the periphery of the fibers intensely (F ig. 4). Uranyl acetate and lead citrate have no such specificity and barely allow the fibers to be resolved (Fig. 5). Glutaraldehyde fixed tissue which has been post osmicated and then incubated in 2 % uranyl acetate overnight at room 211
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temperature (Locke et al., 1971) stains the fibers more than other connective tissue components (Fig. 6) but is less selective. Fig. 7 shows fibers from a connective tissue strand in Calpodes which has been fixed in tannic acid/glutaraldehyde, osmicated and the sections stained with lead citrate. Fibers are obvious in connective tissue from various sources after this treatment such as that below the epidermis, where they were previously unsuspected. From these results it seems probable that elastic fibers may be widespread in insect connective tissue. The staining procedures described do not all show up the same components. The PTA on sections and the uranyl acetate on osmicated tissue react with the surface of the large fibrous component giving density around less
dense fibers. The PTA in particular reacts to give dense amorphous material. The DAB and the tannic acid on the other hand cause the heavy metals (lead or osmium) to react with the whole fiber which appears uniformly dense and rodlike. Presumably the tannic acid cross links a variety of proteins in competition with glutaraldehyde and in particular those of the elastic fibers. These proteins then have free aromatic hydroxyl groups which react with heavy metals. Acknowledgements This work was generously supported by the National Research Council of Canada, Grant no. A6607.
m-matrix c-s-collagen f---elastic fibers h-haemocoel gl-glial cell Figs. 1-6. Adult female locust neural lamella transverse sections. Fig. 7, Calpodes pericardial connective tissue fiber. Fig. I. Glutaraldehyde fixation, DAB reaction without H 2 0 2 , osmication. x 11,600. Figs. 2, 3. As Fig. 1 but section stained with uranyl acetate and lead citrate. Fig. 2, x 11,000. Fig. 3, x 39,000. Fig. 4. Glutaraldehyde fixation, section stained with aqueous PTA. x 39,000. Fig. 5. Glutaraldehyde fixation, section stained with uranyl acetate and lead citrate. x 39,000. Fig. 6. Glutaraldehyde fixation, osmication, tissue stained with uranyl acetate. x 41,000. Fig. 7. Glutaraldehyde and tannic acid fixation, section stained with lead citrate. x 82,000.
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1 J.l
f......
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CD
h m
h m
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ELASTIC FIBERS IN CONNECTIVE TISSUE
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References GREENLEE, T. K., Ross, R. and HARTMAN, J. L. 1966. The fine structure of elastic fibers. J. Cell BioI., 30 (I), 59-71. LOCKE, M., KRISHNAN, N. and McMAHON, J. 1971. A routine method of obtaining high contrast without staining sections. J. Cell BioI., 50, 540-544. LOCKE, M. and HUIE, P. 1972. The fiber components of insect connective tissue. Tissue & Cell, 4 (4), 601-612. RICHARDS, V. A. G. and SCHNEIDER, D. 1958. Uber den komplexen Bau der Membranen des Bindegewebes von Insekten. Z. Naturf., 13 (10), 680-687. Ross, R. and BORNSTEIN, P. 1969. The elastic fiber. 1. The separation and partial characterization of its macromolecular components. J. Cell BioI., 40 (2), 366-381. TILNEY, L. G., BRYAN, J., BUSH, D. J., FUJIWARA, K., MOOSEKER, M. S., MURPHY, D. B. and SNYDER, D. H. (1973). Microtubules: Evidence for 13 protofilaments. J. Cell BioI., 59, 267-275. VINCENT, J. F. and WOOD, S. D. (1972). Mechanisms of abdominal extension during oviposition in Locus/a. Nature, Lond., 235 (5334), 167-168. VINCENT, J. F. V. and PRENTICE, J. H. (1973). Rheological properties of the extensible intersegmental membrane of the adult female locust. J. Mat, Sci., 8, 624-630.