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The use of a tannic acid—glutaraldehyde fixative to visualize gap and tight junctions
JOURNALOF ULTRASTRUCTURERESEARCH50, 185-192 (1975)
The Use of a Tannic Acid-Glutaraldehyde Fixative to Visualize Gap and Tight Junctions B. VAN DEURS
Anatomy Department A, University of Copenhagen, 71, Raadmandsgade, D K 2200 Copenhagen N., Denmark Received April 23, 1974 Fixation of livers from mice with a mixture of tannic acid and glutaraldehyde resulted in an increased density of the cell periphery. Gap and tight junctions were well demonstrated by this method. The gap junctions were filled with a homogeneous, electron-dense precipitate. Faint electron-lucent lines spaced 100 A center-to-center crossed the gaps. In oblique sections of gap junctions an array of round particles was seen. The tight junctions appeared as membrane fusions preventing the passage of the tannic acid from the intercellular clefts to the bile canalieuli. Morphologically, the results obtained by using tannic acid as a tracer are similar to those obtained with lanthanum and ruthenium red, but chemically the tannic acid may act in a different way, thus representing an "alternative" tracer. F i x a t i v e s for e l e c t r o n m i c r o s c o p y containing t a n n i c acid have recently been used to d e m o n s t r a t e t h e s u b s t r u c t u r e of m i c r o t u b u l e s (1, 6, 9), to s t a i n e l a s t i c fibers, t h e i n t e r m e d i a t e l a m i n a of e p i t h e l i a l desm o s o m e s a n d t h e cell c o a t of i n t e s t i n a l m i c r o v i l l i (1), a n d to d e m o n s t r a t e t h e porous s u b s t r u c t u r e of t h e g l o m e r u l a r slit d i a p h r a g m (8). T h e p u r p o s e of t h i s s t u d y was to i n v e s t i g a t e t h e p o s s i b l e a d v a n t a g e s of a t a n n i c a c i d - g l u t a r a l d e h y d e f i x a t i v e , a p p l i e d to t h e t i s s u e e i t h e r b y i m m e r s i o n or by p e r f u s i o n , in e l u c i d a t i n g t h e d i s t r i b u t i o n a n d s t r u c t u r e of i n t e r c e l l u l a r j u n c t i o n s . T h e l i v e r w a s u s e d as t e s t t i s s u e s i n ce b o t h g a p a n d t i g h t j u n c t i o n s p r e v i o u s l y h a v e b e e n s t u d i e d in d e t a i l h e r e (2), a n d also d e s m o s o m e s are p r e s e n t b e t w e e n the hepatocytes.
Immersion fixation procedure. Small pieces of liver from adult BALB/C mice were fixed in these fixatives for 1 hr at room temperature, followed by 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2, for I hr. Control specimens were prepared using 2.5% gtutaraldehyde, pH 7.2, and 1% osmium tetroxide. Perfusion fixation procedure. Adult BALB/C mice were anaesthetized with ether, and perfusion with Hanks balanced salts solution, 38°C, was performed through the left ventricle for 2 rain until complete blood depletion of the vascular system. The mice were then perfused for 5 rain with a TAG fixative containing 4% TA (see above) at a flow rate of 10 ml/min. Small pieces of perfused liver were thereafter fixed in 2.5% glutaraldehyde in 0.2 M buffer, pH 7.2, for 1 hr at room temperature, and postfixed in 1% osmium tetroxide. After fixation, all tissue were block-stained for 1 hr at room temperature in 0.5% uranyl acetate in water, dehydrated in graded alcohols, and embedded in Epon. Sections were studied in a Hitachi HS-8 electron microscope without any further staining, or after staining with lead citrate for 2-5 rain.
MATERIALS AND METHODS RESULTS A "standard" solution of tannic acid (TA) was prepared by dissolving 16% Tannin (Ph.Nord. 63) in Consistent results were o b t a i n e d with 0.2 M Na-cacodylate buffer, pH 7.4, at 50°C. Both the t h e T A G f i x a t i v e s c o n t a i n i n g 4% or 8% T A , standard solution and 8%, 4%, and 2% TA solutions w h i l e t h e r e s u l t s o b t a i n e d w i t h l o w er conprepared at room temperature from the standard solution by diluting it with the buffer, were mixed c e n t r a t i o n s (1% a n d 2%) were c a p r i c i o u s . immediately before fixation with equal volumes of 5% All m i c r o g r a p h s of T A G f i x e d t i s s u e glutaraldehyde in 0.2 M Na-cacodylate buffer, pH s h o w n in t h i s p a p e r are f r o m t h e 4% T A 7.2. The four final tannic acid-glutaraldehyde (TAG) fixatives thus contained 1%, 2%, 4%, or 8% TA and f i x a t i o n , e i t h e r i m m e r s i o n or p e r f u s i o n , 2.5% glutaraldehyde in 0.2 M buffer, and the pH's of a n d t h e s e c t i o n s were all s t a i n e d w i t h l e a d the mixtures were 7.0, 6.8, 6.5, and 6.2, respectively. c i t r a t e . 185 Copyright © 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
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At low magnification the sinusoids and the intercellular spaces between hepatocytes from liver fixed by immersion were clearly visible as dark areas and lines (Fig. 1). The space of Disse in particular was clearly d e m o n s t r a t e d being filled with an e l e c t r o n - d e n s e , f l o c c u l e n t m a t e r i a l between the microvilli (Fig. 1, Ds). In the tissue fixed by perfusion, no flocculent material was seen in the sinusoids or in the space of Disse. The cell periphery (defined as the outer leaflet of the plasma membrane plus associated cell coat substance) of microvilli, endothelial cells, and of blood cells situated in the sinusoids showed an increased density no m a t t e r what fixation procedure was used (Fig. 3). In the bile canaliculi generally no dense material was seen (Fig. 1, Bc2). However, in the peripheral parts of the tissue blocks of immersion-fixed liver the dense, flocculent material was sometimes present in the bile canaliculi, a p h e n o m e n o n due to the immersion fixation procedure (Fig. 1, Bcl), because this was never observed after fixation by perfusion. At higher magnifications the intercellular spaces between adjacent hepatocytes from immersion-fixed tissue were found to be filled with a flocculent material similar to t h a t found, e.g., in the space of Disse, although it was less p r o m i n e n t (Fig. 2). In tissue fixed by perfusion such material was in general only present in the intercellular spaces close to tight junctions (see below) (Figs. 9 and 10). Gap junctions were clearly outlined by both fixation procedures (Figs. 4-6, and 9). T h e y were often situated in connection with "loops" of adjacent h e p a t o c y t e mere-
branes (Fig. 4). The width of the p l a s m a m e m b r a n e was about 75 A, and the overall width of the gap junction was approxim a t e l y 180 ~. The gap between the outer leaflets of adjacent m e m b r a n e s was filled with a homogeneous electron-dense material (in contrast to the flocculent material in the nonjunctional 200 A space of the immersion-fixed tissue). The TA-penet r a t e d gap measured 40-60 ~ in width. T h e intercellular space of the gap junction was thus more clearly visualized t h a n in blockstained routine preparations (Fig. 7), in which the gap often was invisible, and the junction thus appeared p e n t a l a m i n a r (Fig. 8). In oblique sections of the gap junction round particles, about 50 A in diameter, were seen (Fig. 4, GjL). When the junctional m e m b r a n e s were perpendicular to the plane of section faint, electron-lucent lines crossing the gap were visible, These lines were regularly spaced, about 100 center-to-center (Fig. 5). When the junctional m e m b r a n e s were slightly tilted in the plane of section, a transition from the lines to the round particles in the junction was seen. Tight junctions were visualized as membrane fusions sealing the intercellular space between hepatocytes from the bile canaliculi, preventing the entrance of T A into these. Thus, the flocculent, electrondense material was only present on the intercellular space-side of the tight junction (Figs. 10 and 11). In general, no effects of the T A on the organelles of the intact h e p a t o c y t e were observed. However, in occasionally damaged cells there was a pronounced effect on the cytoplasm and the organelles. The
FIG. 1. Low magnification micrograph of a sinusoid surrounded by four hepatocytes. The lumen of the sinuosid is occupied by a Kupffer cell (Kc). In the space of Disse (Ds) and in the intercellular clefts some dense, fiocculent material is present. Two bile canaliculi (Bcl and Bc2) are seen, the first being filled with an electron-dense material, while the other is empty. Arrowsindicate the position of tight junctions sealing the bile canaliculi. Immersion fixation. × 13 000 FIG. 2. A nonjunctional intercellular cleft between two hepatocytes. Note the fiocculent precipitate in the cle~ and the darkly, TA-stained periphery. Immersion fixation. × 160 000 Fro. 3. Part of an erythrocyte from a sinuosid. The cell periphery (arrows) is heavily stained by the TA. Immersion fixation. × 145 000
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cisternae of the endoplasmic reticulum and the perinuclear space were swollen. The outer membrane of mitochondria showed an increase in thickness and density, while the inner membrane and those of the cristae did not (Fig.. 12). Lipid droplets were not stained by the TA (Fig. 12, L). In damaged cells both leaflets of the cell membranes showed increased density (Fig. 13), and the tonofilaments on the cytoplasmic side of desmosomes were heavily stained (Fig. 13, Tf).
rial observed after TAG fixation has not been established in detail. Futaesaku et al. (1) suggested the effect of TA to be due to the formation of complexes between proteins in a colloidal form having positive charges and the TA, which probably is negatively charged. These complexes may bind osmium and/or other heavy metals (e.g., uranium) resulting in the formation of an electron-dense precipitate. Further studies of these problems are being carried out at this laboratory. The cell periphery shows an increased DISCUSSION density after the TAG fixation. This pheBecause of the acidity of tannic acid, a nomenon is likely to be due to the reaction drastic fall in the pH-values of the TAG between TA and glycoproteins or other fixatives was prevented by using 0.2 M membrane associated proteins. The "acbuffer, Four percent TA was preferred tion" of TA is thus different from that of since on the one hand 1% and 2% TA both colloidal and ionic lanthanum and appeared to be less effective than 4% TA, ruthenium red, which are cations and and 8% TA on the other hand did not have probably bind to the carbon hydrate comany advantages to 4% and may introduce ponents of the cell periphery (4, 5, 7). The more artefacts than 4%. It is likely, how- flocculent precipitate seen in the intercelever, that other concentrations would be lular spaces and in the sinusoids of liver optimal with respect to the preservation of fixed by immersion is probably due to the cell periphery in the application of binding between serum proteins and TA, TAG to other tissues. So far, we have had but it facilitates the identification of the equivalent results to those from the liver by intercellular spaces, when the sections are using lymph nodes as test tissue (unpub- studied at low magnification. lished observations). When fixed by imSince the width of the plasma membrane mersion, the tissue blocks must be as small measured about 75 A and the overall width as possible since the TA does not penetrate of the gap junction measured about 180 A, deeply in the tissue. Finally, the uniform- the gap must be about 30 ~. However, ity and purity of the commercial product when permeated with TA the gap measures Tannin may also be questioned. 40-60 A. This inconsistency may be exThe nature of the electron-dense mate- plained by the fact that also the outer FIG. 4. Gap junctional area between two hepatocytes, situated in connection with a loop of the two adjacent cell membranes. When the gap junction is sectioned perpendicular to the adjacent membranes, the intercellular cleft is seen to be filled with an electron-dense material (Gj). In oblique sections of the junction, a pattern of subunits, the gap junction lattice (GjL), is seen. Immersion fixation, x 95 000 FIG. 5. Part of a gap junction showing the faint, electron-lucent lines crossing the gap (arrows). At the left a transition to the gap junction lattice (GjL) is seen. To the right is a part of the nonjunctional intercellular space (Is). Immersion fixation, x 169 000 FIG. 6. Section of a long, straight gap junction showing the homogeneous, electron-dense material in the junctional space. Immersion fixation, x 180 000 Fro. 7. Control preparation (without TA) of a gap junction, in which the intercellular space is visible. Immersion fixation, x 180 000 FIG. 8. Control preparation of an other gap junction where the intercellular space is invisible, and the junction thus appears pentalaminar. Immersion fixation, x 180 000
FIG. 9. Two a d j a c e n t h e p a t o c y t e s from liver fixed by perfusion. T h e n o n j u n c t i o n a l intercellular space (Is) is a l m o s t e m p t y , while the space of t h e gap j u n c t i o n (Gj) is occupied by an electron-dense material. × 100 000 Fro. 10. T i g h t j u n c t i o n (arrow) sealing the intercellular space (Is) from the bile c a n a l i c u l u s (Bc). Note t h e flocculent m a t e r i a l in t h e intercellular space close to the junction. Perfusion fixation. × 92 000 Fro. 11. A m i c r o g r a p h showing the s a m e as Fig. 10, b u t t h e tissue here is fixed by i m m e r s i o n . × 92 000 190
JUNCTION S VISUALIZEDBY TANNIC ACID
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FIC. 12. Part of a damaged hepatocyte. The cisternae of the endoplasmic reticulum (Er) are swollen. Note that the outer membrane of the mitochondrium (M) is heavily stained by the TA (arrows). The lipid droplet (L) is, on the other hand, not stained. Immersion fixation. × 57 000 FIa. 13. A desmosome between two damaged hepatocytes. The tonofilaments (Tf) are heavily stained by the TA, and so are both the outer and the inner leaflets of the hepatocyte membranes. This is particularly evident within the desmosome. Immersion fixation. × 180 000 leaflets of the junctional m e m b r a n e s are T A - s t a i n e d and thus impossible to discern from the homogeneous, electron-dense material in the junctional space. A lattice of hexagonal or polygonal arranged subunits with a center-to-center spacing of a b o u t 100 ~ in the gap junction has been shown several times by different methods, e.g., l a n t h a n u m (see 2, 7) and freeze-cleaving (see 2, 3). The faint electron-lucent lines crossing the gap junction
are likely to represent the subunits of a "gap junction lattice" (Fig. 4, GjL), which m a y facilitate electrotonic coupling between cells as well as intercellular adhesion (for discussion, see 2). Since the subunits are not directly stained by the TA, b u t are negatively outlined, and the lipid droplets of d a m a g e d cells also are not TA-stained, one m a y speculate t h a t they are composed, at least partly, of lipids, probably bridging the lipid bilayers of the two adjacent unit
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membranes. Goodenough and Revel (2) also found support for this by "removing" the subunits of the gap junction lattice with acetone. It is concluded that the TAG fixation seems to represent an applicable tracer method alternative to lanthanum and ruthenium red. REFERENCES 1. FUTAESAKU,Y., MIZUHIRA,V., AND NAKAMURA,H., Proc. Int. Congr. Histochem. Cytochem. 4, 155 (1972).
2. GOODENOUGH,D. A,, ANDREVEL, J. P., J. Cell Biol. 45, 272 (1970). 3. KREUTZmER,G. O., Proc. Elec. Micro. Soc. Amer. 26, 234 (1968). 4. Lc~'r, J. H., Anat. Rec. 171, 347 (1971). 5. LUFT, J. H., Anat. Rec. 171, 369 (1971). 6. MIzunmA, V., AND FUTAESAKU, Y., Proc. Elec. Micro. Soc. Amer. 29, 494 (1971). 7. REVEL, J. P., AND KARNOVSKV,M. J., J. Cell Biol. 33, C7 (1967). 8. RODEWALD,R., ANDKARNOVSKY,M. J., J. Cell Biol. 60, 423 (1974). 9. TILNEY, L. G., BRYAN, J., BUSH, D. J., FUJIWARA, K., MOOSEKER, M. S., MUaPHV, D. B., AND SNYDER, D. H., J. Cell Biol. 59, 267 (1973).
The Use of a Tannic Acid-Glutaraldehyde Fixative to Visualize Gap and Tight Junctions B. VAN DEURS
Anatomy Department A, University of Copenhagen, 71, Raadmandsgade, D K 2200 Copenhagen N., Denmark Received April 23, 1974 Fixation of livers from mice with a mixture of tannic acid and glutaraldehyde resulted in an increased density of the cell periphery. Gap and tight junctions were well demonstrated by this method. The gap junctions were filled with a homogeneous, electron-dense precipitate. Faint electron-lucent lines spaced 100 A center-to-center crossed the gaps. In oblique sections of gap junctions an array of round particles was seen. The tight junctions appeared as membrane fusions preventing the passage of the tannic acid from the intercellular clefts to the bile canalieuli. Morphologically, the results obtained by using tannic acid as a tracer are similar to those obtained with lanthanum and ruthenium red, but chemically the tannic acid may act in a different way, thus representing an "alternative" tracer. F i x a t i v e s for e l e c t r o n m i c r o s c o p y containing t a n n i c acid have recently been used to d e m o n s t r a t e t h e s u b s t r u c t u r e of m i c r o t u b u l e s (1, 6, 9), to s t a i n e l a s t i c fibers, t h e i n t e r m e d i a t e l a m i n a of e p i t h e l i a l desm o s o m e s a n d t h e cell c o a t of i n t e s t i n a l m i c r o v i l l i (1), a n d to d e m o n s t r a t e t h e porous s u b s t r u c t u r e of t h e g l o m e r u l a r slit d i a p h r a g m (8). T h e p u r p o s e of t h i s s t u d y was to i n v e s t i g a t e t h e p o s s i b l e a d v a n t a g e s of a t a n n i c a c i d - g l u t a r a l d e h y d e f i x a t i v e , a p p l i e d to t h e t i s s u e e i t h e r b y i m m e r s i o n or by p e r f u s i o n , in e l u c i d a t i n g t h e d i s t r i b u t i o n a n d s t r u c t u r e of i n t e r c e l l u l a r j u n c t i o n s . T h e l i v e r w a s u s e d as t e s t t i s s u e s i n ce b o t h g a p a n d t i g h t j u n c t i o n s p r e v i o u s l y h a v e b e e n s t u d i e d in d e t a i l h e r e (2), a n d also d e s m o s o m e s are p r e s e n t b e t w e e n the hepatocytes.
Immersion fixation procedure. Small pieces of liver from adult BALB/C mice were fixed in these fixatives for 1 hr at room temperature, followed by 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2, for I hr. Control specimens were prepared using 2.5% gtutaraldehyde, pH 7.2, and 1% osmium tetroxide. Perfusion fixation procedure. Adult BALB/C mice were anaesthetized with ether, and perfusion with Hanks balanced salts solution, 38°C, was performed through the left ventricle for 2 rain until complete blood depletion of the vascular system. The mice were then perfused for 5 rain with a TAG fixative containing 4% TA (see above) at a flow rate of 10 ml/min. Small pieces of perfused liver were thereafter fixed in 2.5% glutaraldehyde in 0.2 M buffer, pH 7.2, for 1 hr at room temperature, and postfixed in 1% osmium tetroxide. After fixation, all tissue were block-stained for 1 hr at room temperature in 0.5% uranyl acetate in water, dehydrated in graded alcohols, and embedded in Epon. Sections were studied in a Hitachi HS-8 electron microscope without any further staining, or after staining with lead citrate for 2-5 rain.
MATERIALS AND METHODS RESULTS A "standard" solution of tannic acid (TA) was prepared by dissolving 16% Tannin (Ph.Nord. 63) in Consistent results were o b t a i n e d with 0.2 M Na-cacodylate buffer, pH 7.4, at 50°C. Both the t h e T A G f i x a t i v e s c o n t a i n i n g 4% or 8% T A , standard solution and 8%, 4%, and 2% TA solutions w h i l e t h e r e s u l t s o b t a i n e d w i t h l o w er conprepared at room temperature from the standard solution by diluting it with the buffer, were mixed c e n t r a t i o n s (1% a n d 2%) were c a p r i c i o u s . immediately before fixation with equal volumes of 5% All m i c r o g r a p h s of T A G f i x e d t i s s u e glutaraldehyde in 0.2 M Na-cacodylate buffer, pH s h o w n in t h i s p a p e r are f r o m t h e 4% T A 7.2. The four final tannic acid-glutaraldehyde (TAG) fixatives thus contained 1%, 2%, 4%, or 8% TA and f i x a t i o n , e i t h e r i m m e r s i o n or p e r f u s i o n , 2.5% glutaraldehyde in 0.2 M buffer, and the pH's of a n d t h e s e c t i o n s were all s t a i n e d w i t h l e a d the mixtures were 7.0, 6.8, 6.5, and 6.2, respectively. c i t r a t e . 185 Copyright © 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
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At low magnification the sinusoids and the intercellular spaces between hepatocytes from liver fixed by immersion were clearly visible as dark areas and lines (Fig. 1). The space of Disse in particular was clearly d e m o n s t r a t e d being filled with an e l e c t r o n - d e n s e , f l o c c u l e n t m a t e r i a l between the microvilli (Fig. 1, Ds). In the tissue fixed by perfusion, no flocculent material was seen in the sinusoids or in the space of Disse. The cell periphery (defined as the outer leaflet of the plasma membrane plus associated cell coat substance) of microvilli, endothelial cells, and of blood cells situated in the sinusoids showed an increased density no m a t t e r what fixation procedure was used (Fig. 3). In the bile canaliculi generally no dense material was seen (Fig. 1, Bc2). However, in the peripheral parts of the tissue blocks of immersion-fixed liver the dense, flocculent material was sometimes present in the bile canaliculi, a p h e n o m e n o n due to the immersion fixation procedure (Fig. 1, Bcl), because this was never observed after fixation by perfusion. At higher magnifications the intercellular spaces between adjacent hepatocytes from immersion-fixed tissue were found to be filled with a flocculent material similar to t h a t found, e.g., in the space of Disse, although it was less p r o m i n e n t (Fig. 2). In tissue fixed by perfusion such material was in general only present in the intercellular spaces close to tight junctions (see below) (Figs. 9 and 10). Gap junctions were clearly outlined by both fixation procedures (Figs. 4-6, and 9). T h e y were often situated in connection with "loops" of adjacent h e p a t o c y t e mere-
branes (Fig. 4). The width of the p l a s m a m e m b r a n e was about 75 A, and the overall width of the gap junction was approxim a t e l y 180 ~. The gap between the outer leaflets of adjacent m e m b r a n e s was filled with a homogeneous electron-dense material (in contrast to the flocculent material in the nonjunctional 200 A space of the immersion-fixed tissue). The TA-penet r a t e d gap measured 40-60 ~ in width. T h e intercellular space of the gap junction was thus more clearly visualized t h a n in blockstained routine preparations (Fig. 7), in which the gap often was invisible, and the junction thus appeared p e n t a l a m i n a r (Fig. 8). In oblique sections of the gap junction round particles, about 50 A in diameter, were seen (Fig. 4, GjL). When the junctional m e m b r a n e s were perpendicular to the plane of section faint, electron-lucent lines crossing the gap were visible, These lines were regularly spaced, about 100 center-to-center (Fig. 5). When the junctional m e m b r a n e s were slightly tilted in the plane of section, a transition from the lines to the round particles in the junction was seen. Tight junctions were visualized as membrane fusions sealing the intercellular space between hepatocytes from the bile canaliculi, preventing the entrance of T A into these. Thus, the flocculent, electrondense material was only present on the intercellular space-side of the tight junction (Figs. 10 and 11). In general, no effects of the T A on the organelles of the intact h e p a t o c y t e were observed. However, in occasionally damaged cells there was a pronounced effect on the cytoplasm and the organelles. The
FIG. 1. Low magnification micrograph of a sinusoid surrounded by four hepatocytes. The lumen of the sinuosid is occupied by a Kupffer cell (Kc). In the space of Disse (Ds) and in the intercellular clefts some dense, fiocculent material is present. Two bile canaliculi (Bcl and Bc2) are seen, the first being filled with an electron-dense material, while the other is empty. Arrowsindicate the position of tight junctions sealing the bile canaliculi. Immersion fixation. × 13 000 FIG. 2. A nonjunctional intercellular cleft between two hepatocytes. Note the fiocculent precipitate in the cle~ and the darkly, TA-stained periphery. Immersion fixation. × 160 000 Fro. 3. Part of an erythrocyte from a sinuosid. The cell periphery (arrows) is heavily stained by the TA. Immersion fixation. × 145 000
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cisternae of the endoplasmic reticulum and the perinuclear space were swollen. The outer membrane of mitochondria showed an increase in thickness and density, while the inner membrane and those of the cristae did not (Fig.. 12). Lipid droplets were not stained by the TA (Fig. 12, L). In damaged cells both leaflets of the cell membranes showed increased density (Fig. 13), and the tonofilaments on the cytoplasmic side of desmosomes were heavily stained (Fig. 13, Tf).
rial observed after TAG fixation has not been established in detail. Futaesaku et al. (1) suggested the effect of TA to be due to the formation of complexes between proteins in a colloidal form having positive charges and the TA, which probably is negatively charged. These complexes may bind osmium and/or other heavy metals (e.g., uranium) resulting in the formation of an electron-dense precipitate. Further studies of these problems are being carried out at this laboratory. The cell periphery shows an increased DISCUSSION density after the TAG fixation. This pheBecause of the acidity of tannic acid, a nomenon is likely to be due to the reaction drastic fall in the pH-values of the TAG between TA and glycoproteins or other fixatives was prevented by using 0.2 M membrane associated proteins. The "acbuffer, Four percent TA was preferred tion" of TA is thus different from that of since on the one hand 1% and 2% TA both colloidal and ionic lanthanum and appeared to be less effective than 4% TA, ruthenium red, which are cations and and 8% TA on the other hand did not have probably bind to the carbon hydrate comany advantages to 4% and may introduce ponents of the cell periphery (4, 5, 7). The more artefacts than 4%. It is likely, how- flocculent precipitate seen in the intercelever, that other concentrations would be lular spaces and in the sinusoids of liver optimal with respect to the preservation of fixed by immersion is probably due to the cell periphery in the application of binding between serum proteins and TA, TAG to other tissues. So far, we have had but it facilitates the identification of the equivalent results to those from the liver by intercellular spaces, when the sections are using lymph nodes as test tissue (unpub- studied at low magnification. lished observations). When fixed by imSince the width of the plasma membrane mersion, the tissue blocks must be as small measured about 75 A and the overall width as possible since the TA does not penetrate of the gap junction measured about 180 A, deeply in the tissue. Finally, the uniform- the gap must be about 30 ~. However, ity and purity of the commercial product when permeated with TA the gap measures Tannin may also be questioned. 40-60 A. This inconsistency may be exThe nature of the electron-dense mate- plained by the fact that also the outer FIG. 4. Gap junctional area between two hepatocytes, situated in connection with a loop of the two adjacent cell membranes. When the gap junction is sectioned perpendicular to the adjacent membranes, the intercellular cleft is seen to be filled with an electron-dense material (Gj). In oblique sections of the junction, a pattern of subunits, the gap junction lattice (GjL), is seen. Immersion fixation, x 95 000 FIG. 5. Part of a gap junction showing the faint, electron-lucent lines crossing the gap (arrows). At the left a transition to the gap junction lattice (GjL) is seen. To the right is a part of the nonjunctional intercellular space (Is). Immersion fixation, x 169 000 FIG. 6. Section of a long, straight gap junction showing the homogeneous, electron-dense material in the junctional space. Immersion fixation, x 180 000 Fro. 7. Control preparation (without TA) of a gap junction, in which the intercellular space is visible. Immersion fixation, x 180 000 FIG. 8. Control preparation of an other gap junction where the intercellular space is invisible, and the junction thus appears pentalaminar. Immersion fixation, x 180 000
FIG. 9. Two a d j a c e n t h e p a t o c y t e s from liver fixed by perfusion. T h e n o n j u n c t i o n a l intercellular space (Is) is a l m o s t e m p t y , while the space of t h e gap j u n c t i o n (Gj) is occupied by an electron-dense material. × 100 000 Fro. 10. T i g h t j u n c t i o n (arrow) sealing the intercellular space (Is) from the bile c a n a l i c u l u s (Bc). Note t h e flocculent m a t e r i a l in t h e intercellular space close to the junction. Perfusion fixation. × 92 000 Fro. 11. A m i c r o g r a p h showing the s a m e as Fig. 10, b u t t h e tissue here is fixed by i m m e r s i o n . × 92 000 190
JUNCTION S VISUALIZEDBY TANNIC ACID
191
FIC. 12. Part of a damaged hepatocyte. The cisternae of the endoplasmic reticulum (Er) are swollen. Note that the outer membrane of the mitochondrium (M) is heavily stained by the TA (arrows). The lipid droplet (L) is, on the other hand, not stained. Immersion fixation. × 57 000 FIa. 13. A desmosome between two damaged hepatocytes. The tonofilaments (Tf) are heavily stained by the TA, and so are both the outer and the inner leaflets of the hepatocyte membranes. This is particularly evident within the desmosome. Immersion fixation. × 180 000 leaflets of the junctional m e m b r a n e s are T A - s t a i n e d and thus impossible to discern from the homogeneous, electron-dense material in the junctional space. A lattice of hexagonal or polygonal arranged subunits with a center-to-center spacing of a b o u t 100 ~ in the gap junction has been shown several times by different methods, e.g., l a n t h a n u m (see 2, 7) and freeze-cleaving (see 2, 3). The faint electron-lucent lines crossing the gap junction
are likely to represent the subunits of a "gap junction lattice" (Fig. 4, GjL), which m a y facilitate electrotonic coupling between cells as well as intercellular adhesion (for discussion, see 2). Since the subunits are not directly stained by the TA, b u t are negatively outlined, and the lipid droplets of d a m a g e d cells also are not TA-stained, one m a y speculate t h a t they are composed, at least partly, of lipids, probably bridging the lipid bilayers of the two adjacent unit
192
B. VAN DEURS
membranes. Goodenough and Revel (2) also found support for this by "removing" the subunits of the gap junction lattice with acetone. It is concluded that the TAG fixation seems to represent an applicable tracer method alternative to lanthanum and ruthenium red. REFERENCES 1. FUTAESAKU,Y., MIZUHIRA,V., AND NAKAMURA,H., Proc. Int. Congr. Histochem. Cytochem. 4, 155 (1972).
2. GOODENOUGH,D. A,, ANDREVEL, J. P., J. Cell Biol. 45, 272 (1970). 3. KREUTZmER,G. O., Proc. Elec. Micro. Soc. Amer. 26, 234 (1968). 4. Lc~'r, J. H., Anat. Rec. 171, 347 (1971). 5. LUFT, J. H., Anat. Rec. 171, 369 (1971). 6. MIzunmA, V., AND FUTAESAKU, Y., Proc. Elec. Micro. Soc. Amer. 29, 494 (1971). 7. REVEL, J. P., AND KARNOVSKV,M. J., J. Cell Biol. 33, C7 (1967). 8. RODEWALD,R., ANDKARNOVSKY,M. J., J. Cell Biol. 60, 423 (1974). 9. TILNEY, L. G., BRYAN, J., BUSH, D. J., FUJIWARA, K., MOOSEKER, M. S., MUaPHV, D. B., AND SNYDER, D. H., J. Cell Biol. 59, 267 (1973).