- Email: [email protected]
Staining of transverse tubular system of skeletal muscle by tannic acid—glutaraldehyde fixation
JOURNAL OF ULTRASTRUCTURE RESEARCH 58, 1 6 2 - 1 6 5
(1977)
Staining of Transverse Tubular System of Skeletal Muscle by Tannic Acid-Glutaraldehyde Fixation 1 EDUARDO BONILLA
Department of Neurology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104 Received June 4, 1976, and in revised form, August 19, 1976 Fixation by immersion of skeletal muscle with a combination of 8% tannic acid and 2.5% glutaraldehyde revealed a prominent s t a i n i n g of t h e junctional "T" system, cell surface and lameIlar structures a t the cell periphery in u n d a m a g e d muscle cells. The results revealed by this technique are similar to those obtained w i t h r u t h e n i u m red and l a n t h a n u m . However, the preferential increase in electron density at the junctional "T" system may indicate the presence of a special pool of proteins at this site. The exact chemical basis for this observation remains to be elucidated.
The use of fixatives for electron microscopy containing tannic acid was introduced by Mizuhira and Futaesaku (6) and Futaesaku et al. (2). This technique has been previously applied for the study of the substructure of microtubules (2, 6, 9) and for the demonstration of the porous substructure of the glomerular slit diaphragm (7). In more recent work, it has been successfully applied for the visualization of gap and tight junctions in liver cells (10). The purpose of this communication is to report the staining of the transverse tubular CT") system of intact skeletal muscle cells by tannic acid glutaraldehyde fixation. The staining pattern revealed a preferential increase in electron density at the junctional "T~' system. In addition staining of the celI surface and the appearance of lamellar structures at the cell periphery were also observed. MATERIALS AND METHODS U n d e r general anesthesia portions of rabbits adductor m a g n u s were obtained. Tannic acid-glutaraldehyde (TAG): Bundles of 30-50 fibers were removed at rest l e n g t h in a Ushaped muscle clamp and fixed immediately in 2.5% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2) containing 4 and 8% tannic acid (Sigma Chemical Co. St. Louis, Mo.) The final pH of the mixtures was 1 Supported by a g r a n t from the Muscular Dystrophy Association of America, Inc. and by PHS G r a n t NS-08075.
adjusted to 7.2 with 1 N NaOH) for 1 h r (10). After fixation the specimens were released from the clamps and briefly rinsed in buffer. The most external fibers were cut into small blocks and postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2) for 1 hr. The blocks were dehydrated in serial alcohols and embedded in Epon. The embedded m a t e r i a l was sectioned w i t h a diamond knife mounted on an LKB ultramicrotome. T h i n sections were picked up on coated 300 mesh grids. The sections were stained with lead citrate for 5 min. The m a t e r i a l was examined a n d micrographed in a n AEI-6B Electron Microscope operating at 60 kV. RESULTS AND DISCUSSION
T h e best results were obtained with TAG fixatives containing 8% tannic acid and the findings were consistently reproduced in several preparations. All illustrations of TAG fixed skeletal muscle shown in this report are from 8% tannic acid fixation. At low magnifications the plasma membrane, basement membrane and caveolae showed an increase in electron density. The collagen between muscle fibers was heavily stained (Fig. 1). At higher magnifications the outer leaflet of the plasma membrane showed discrete electron dense deposits and floculent electron dense material was associated with the basement membrane (Fig. 2). The "T" system was labeled with electron dense material which penetrated
162 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
"T" SYSTEM STAINING BY TANNIC ACID
FIG. 1. The cell periphery, subcellular components do not Fio. 2. High magnification the outer leaflet of the plasma
163
caveolae and the "T" system show a n increase in electron density. Other show increase in electron density, x 15 000. micrograph of the cell surface. Electron dense deposits are associated with m e m b r a n e and with the b a s e m e n t membrane. × 60 000.
FIG. 3. Intermediate magnification micrograph showing the preferential staining at the junctional "T" system (arrows). x 25 000. FIG. 4. Enlargement of the portion outlined by ink in Fig. 3 illustrating the staining of the junctional ~T" system (arrows) which contrast with the nonjunctional ~T" system (arrowheads). x 90 000. Fro. 5. High magnification micrograph showing a longitudinally oriented triad and portions of longitudinal ~T" system. A straight layer of electron dense material is seen at the junctional ~'T" system (arrows) where as the nonjunctional ~'T"system reveals diffuse and scattered electron dense deposits (arrowheads). x 50 000. FIG. 6. A lamellar structure at the cell periphery. These appear continuous with the plasma membrane. x 60 000. 164
"T" SYSTEM STAINING BY TANNIC ACID
165
deeply into the cells and appeared more the TAG fixation in intact skeletal muscle concentrated at the junctional "T" system cells is similar to those obtained with ru(Fig. 3). At higher magnifications and thenium red and lanthanum (1, 4, 5, 11). when the membranes were perpendicular However, the preferential staining of the to the plane of section the junctional "T" junctional ~T" system with TAG fixation system was stained in a regular fashion may indicate the presence of a special pool appearing as a straight layer of electron of proteins at this location. If so, these dense material. In sharp contrast the non- proteins seem to be located at the site in junctional ~T" system showed irregular which the action potential is transmitted and diffuse electron dense deposits (Figs. 4 to the sarcoplasmic reticulum and it is possible that they might be playing a role and 5). Another important feature of TAG fixa- in the early events of excitation-contraction of skeletal muscle was the appearance tion coupling (8). Further cytochemical of lamellar bodies at the cell periphery. •work specifically designed for the localizaSome of them were quite large and con- tion of glycoproteins at the junctional ~T" sisted of concentric lamellae with a regu- system is under way in an effort to reprolar spacing of about 70/~ (Fig. 6). These duce the results obtained with the TAG structures sometimes appeared continuous fixation method. In summary, TAG fixation of skeletal with the plasma membrane, suggesting they may represent membranous material muscle showed a prominent staining of the of plasma membrane origin organized into junctional "T" system, cell surface and lalamellae by the TAG fixation. It should be mellar structures in undamaged muscle noted that these lamellar structures have cells. The precise chemical basis for these previously been seen in skeletal muscle observations remains to be established. exposed to ruthenium red (3,4). The author wishes to thank Dr. Donald L. SchotIn general, no staining of other cytoplas- land for critical reading of the manuscript and Miss mic organelles of intact muscle cells was Belen Aldover for her skillful technical assistance. observed. However, in damaged cells the REFERENCES labeling of the ~T" system as described 1. FAHIMI, H. D., AND COTRAN, R. S., Amer. J. above was not seen but staining of intraPathol. 62, 143 (1971). cellular components was noted and these 2. FUTAESAKU,Y., MIZUHIRA,V., AND NAKAMURA, H., Proc. Int. Congr. Histochem. Cytochem. 4, patterns are under investigation in this 155 (1972). laboratory. 3. HOWELL, J. N., J. CellBiol. 62, 242 (1974). The exact nature of the electron dense 4. LUFT, Z. H., Anat. Rec. 171, 369 (1971). deposits observed after TAG fixation has 5. LUFF, A. R., ANDATWOOD,H. L . , J . CellBiol. 51, not been fully elucidated. Futaesaku et al. 369 (1971). 6. MIZUHIRA, V., AND FUTAESAKU,Y., Proc. Elec. (2) and Van Deurs (10) have suggested Micro. Soc. Amer. 29, 494 (1971). that ~the effect of tannic acid may be due 7. RODEWALD,R., AND KARNOVSI~Y,M. J., J. Cell to formation of complexes between proBiol. 60~ 423 (1974). teins in a colloidal form having positive 8. SANDOW,A., Pharmacol. Rev. 17, 265 (1965). 9. TILNEY, L. G., BRYAN, J., BUSH, D. J., FUJIcharges and the tannic acid which probaWARA, K., MOOSEKER, M. S., MURPHY, D. B., bly is negatively charged. These comAND SNYDER,D. H . , J . CellBiol. 59, 267 (1973). plexes may bind osmium and/or heavy 10. VAN DEURS, B., J. Ultrastruct. Res. 50, 185 metals resulting in the formation of the (1975). electron dense material" (2, 10). 11. WAUGH, R. A., SPRAY,T. L., AND SOMMER,J. R., J. Cell Biol. 59, 254 (1973). The staining pattern brought about by
(1977)
Staining of Transverse Tubular System of Skeletal Muscle by Tannic Acid-Glutaraldehyde Fixation 1 EDUARDO BONILLA
Department of Neurology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104 Received June 4, 1976, and in revised form, August 19, 1976 Fixation by immersion of skeletal muscle with a combination of 8% tannic acid and 2.5% glutaraldehyde revealed a prominent s t a i n i n g of t h e junctional "T" system, cell surface and lameIlar structures a t the cell periphery in u n d a m a g e d muscle cells. The results revealed by this technique are similar to those obtained w i t h r u t h e n i u m red and l a n t h a n u m . However, the preferential increase in electron density at the junctional "T" system may indicate the presence of a special pool of proteins at this site. The exact chemical basis for this observation remains to be elucidated.
The use of fixatives for electron microscopy containing tannic acid was introduced by Mizuhira and Futaesaku (6) and Futaesaku et al. (2). This technique has been previously applied for the study of the substructure of microtubules (2, 6, 9) and for the demonstration of the porous substructure of the glomerular slit diaphragm (7). In more recent work, it has been successfully applied for the visualization of gap and tight junctions in liver cells (10). The purpose of this communication is to report the staining of the transverse tubular CT") system of intact skeletal muscle cells by tannic acid glutaraldehyde fixation. The staining pattern revealed a preferential increase in electron density at the junctional "T~' system. In addition staining of the celI surface and the appearance of lamellar structures at the cell periphery were also observed. MATERIALS AND METHODS U n d e r general anesthesia portions of rabbits adductor m a g n u s were obtained. Tannic acid-glutaraldehyde (TAG): Bundles of 30-50 fibers were removed at rest l e n g t h in a Ushaped muscle clamp and fixed immediately in 2.5% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2) containing 4 and 8% tannic acid (Sigma Chemical Co. St. Louis, Mo.) The final pH of the mixtures was 1 Supported by a g r a n t from the Muscular Dystrophy Association of America, Inc. and by PHS G r a n t NS-08075.
adjusted to 7.2 with 1 N NaOH) for 1 h r (10). After fixation the specimens were released from the clamps and briefly rinsed in buffer. The most external fibers were cut into small blocks and postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2) for 1 hr. The blocks were dehydrated in serial alcohols and embedded in Epon. The embedded m a t e r i a l was sectioned w i t h a diamond knife mounted on an LKB ultramicrotome. T h i n sections were picked up on coated 300 mesh grids. The sections were stained with lead citrate for 5 min. The m a t e r i a l was examined a n d micrographed in a n AEI-6B Electron Microscope operating at 60 kV. RESULTS AND DISCUSSION
T h e best results were obtained with TAG fixatives containing 8% tannic acid and the findings were consistently reproduced in several preparations. All illustrations of TAG fixed skeletal muscle shown in this report are from 8% tannic acid fixation. At low magnifications the plasma membrane, basement membrane and caveolae showed an increase in electron density. The collagen between muscle fibers was heavily stained (Fig. 1). At higher magnifications the outer leaflet of the plasma membrane showed discrete electron dense deposits and floculent electron dense material was associated with the basement membrane (Fig. 2). The "T" system was labeled with electron dense material which penetrated
162 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
"T" SYSTEM STAINING BY TANNIC ACID
FIG. 1. The cell periphery, subcellular components do not Fio. 2. High magnification the outer leaflet of the plasma
163
caveolae and the "T" system show a n increase in electron density. Other show increase in electron density, x 15 000. micrograph of the cell surface. Electron dense deposits are associated with m e m b r a n e and with the b a s e m e n t membrane. × 60 000.
FIG. 3. Intermediate magnification micrograph showing the preferential staining at the junctional "T" system (arrows). x 25 000. FIG. 4. Enlargement of the portion outlined by ink in Fig. 3 illustrating the staining of the junctional ~T" system (arrows) which contrast with the nonjunctional ~T" system (arrowheads). x 90 000. Fro. 5. High magnification micrograph showing a longitudinally oriented triad and portions of longitudinal ~T" system. A straight layer of electron dense material is seen at the junctional ~'T" system (arrows) where as the nonjunctional ~'T"system reveals diffuse and scattered electron dense deposits (arrowheads). x 50 000. FIG. 6. A lamellar structure at the cell periphery. These appear continuous with the plasma membrane. x 60 000. 164
"T" SYSTEM STAINING BY TANNIC ACID
165
deeply into the cells and appeared more the TAG fixation in intact skeletal muscle concentrated at the junctional "T" system cells is similar to those obtained with ru(Fig. 3). At higher magnifications and thenium red and lanthanum (1, 4, 5, 11). when the membranes were perpendicular However, the preferential staining of the to the plane of section the junctional "T" junctional ~T" system with TAG fixation system was stained in a regular fashion may indicate the presence of a special pool appearing as a straight layer of electron of proteins at this location. If so, these dense material. In sharp contrast the non- proteins seem to be located at the site in junctional ~T" system showed irregular which the action potential is transmitted and diffuse electron dense deposits (Figs. 4 to the sarcoplasmic reticulum and it is possible that they might be playing a role and 5). Another important feature of TAG fixa- in the early events of excitation-contraction of skeletal muscle was the appearance tion coupling (8). Further cytochemical of lamellar bodies at the cell periphery. •work specifically designed for the localizaSome of them were quite large and con- tion of glycoproteins at the junctional ~T" sisted of concentric lamellae with a regu- system is under way in an effort to reprolar spacing of about 70/~ (Fig. 6). These duce the results obtained with the TAG structures sometimes appeared continuous fixation method. In summary, TAG fixation of skeletal with the plasma membrane, suggesting they may represent membranous material muscle showed a prominent staining of the of plasma membrane origin organized into junctional "T" system, cell surface and lalamellae by the TAG fixation. It should be mellar structures in undamaged muscle noted that these lamellar structures have cells. The precise chemical basis for these previously been seen in skeletal muscle observations remains to be established. exposed to ruthenium red (3,4). The author wishes to thank Dr. Donald L. SchotIn general, no staining of other cytoplas- land for critical reading of the manuscript and Miss mic organelles of intact muscle cells was Belen Aldover for her skillful technical assistance. observed. However, in damaged cells the REFERENCES labeling of the ~T" system as described 1. FAHIMI, H. D., AND COTRAN, R. S., Amer. J. above was not seen but staining of intraPathol. 62, 143 (1971). cellular components was noted and these 2. FUTAESAKU,Y., MIZUHIRA,V., AND NAKAMURA, H., Proc. Int. Congr. Histochem. Cytochem. 4, patterns are under investigation in this 155 (1972). laboratory. 3. HOWELL, J. N., J. CellBiol. 62, 242 (1974). The exact nature of the electron dense 4. LUFT, Z. H., Anat. Rec. 171, 369 (1971). deposits observed after TAG fixation has 5. LUFF, A. R., ANDATWOOD,H. L . , J . CellBiol. 51, not been fully elucidated. Futaesaku et al. 369 (1971). 6. MIZUHIRA, V., AND FUTAESAKU,Y., Proc. Elec. (2) and Van Deurs (10) have suggested Micro. Soc. Amer. 29, 494 (1971). that ~the effect of tannic acid may be due 7. RODEWALD,R., AND KARNOVSI~Y,M. J., J. Cell to formation of complexes between proBiol. 60~ 423 (1974). teins in a colloidal form having positive 8. SANDOW,A., Pharmacol. Rev. 17, 265 (1965). 9. TILNEY, L. G., BRYAN, J., BUSH, D. J., FUJIcharges and the tannic acid which probaWARA, K., MOOSEKER, M. S., MURPHY, D. B., bly is negatively charged. These comAND SNYDER,D. H . , J . CellBiol. 59, 267 (1973). plexes may bind osmium and/or heavy 10. VAN DEURS, B., J. Ultrastruct. Res. 50, 185 metals resulting in the formation of the (1975). electron dense material" (2, 10). 11. WAUGH, R. A., SPRAY,T. L., AND SOMMER,J. R., J. Cell Biol. 59, 254 (1973). The staining pattern brought about by