- Email: [email protected]
Effects of different fixatives on the ultrastructure of the developing proximal tubule in the rat kidney
JOURNAL OF ULTRASTRUCTURE RESEARCH 5l, 140-151 (1975)
Effects of Different Fixatives on the Ultrastructure of the Developing Proximal Tubule in the Rat Kidney LARS LARSSON
Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark Received August 13, 1974; accepted September 23, 1974 The fine structure of developing renal tubule cells was evaluated following fixation by different fixatives (osmium tetroxide, glutaraldehyde, formaldehyde, and different combinations of aldehydes) appIied either by vascular perfusion, immersion or dripping on the kidney surface. Vascular perfusion with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer gave a uniform preservation of the developing renal cortex and facilitated a detailed comparison between different developmental stages of the proximal tubule in the same animal.
MATERIALS AND METHODS
INTRODUCTION
Embryonic tissues are generally considered to be difficult to fix for electron microscopy (7, 9, 10, 12, I5, 40) and in the case of renal tissue (8) particular difficulty has been encountered in obtaining uniform preservation throughout the cortex which contains different developmental stages of the nephron (19). In previous studies of developing kidney tubules, the tissue has been fixed by immersion (2, 8, 20, 24, 26, 31, 33, 48, 49, 51-54). However, as pointed out by Clark (8), this procedure results in fixation of different quality at different levels of the renal cortex and makes it very difficult to analyze the fine structure of tubules of different developmental stages. This paper reports results obtained with various fixatives applied in different ways and evaluates the results with particular emphasis on the developing proximal tubule. The conclusions from this work have served as the basis for a series ofultrastructural studies of the developing proximal tubule (28, 29, 32). The definition of the developmental stages of the proximal tubule is the same as presented in detail in the adjoining paper
(28). 140 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
Fifty-six male Wistar rats from 4 to 48 hours old were used. Each experimental group consisted of three to seven rats. The rats were anaesthetized by intraperitoneal injections of sodium pentobarbital (Nembutal, Abbotts Laboratories) of 50 mg/kg body wt. Composition of fixatives. Glutaraldehyde (25%, Merck) was purified by shaking with charcoal (13). The concentration of the glutaraldehyde was determined by measuring the freezing point depression of the solution with an Advanced Osmometer (Model 3L) and taking 1% glutaraldehyde to have an osmolality of 100 mOsm/kg H20 (13). Formaldehyde solutions were prepared as described by Pease (42). Each of the following fixatives was used on one group of animals: (1) 6% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, 828 mOsm/kg H20; (2) 6% glutaraldehyde in 0.01 M sodium cacodylate buffer, pH 7.2, 616 mOsm/kg H20; (3) 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, 506 mOsm/kg H20; (4) 1% osmium tetroxide in 0.029 M Veronal acetate buffer made isotonic with salts (47), pH 7.2, 349 mOsm/kg H20; (5) 4% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, 1450 mOsm/kg H20; (6) 5% glutaraldehyde and 4% formaldehyde in 0.1 M sodium cacodylate buffer (23), pH 7.2, 1990 mOsm/kg H20; (7) 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (23), pH 7.2, 1080 mOsm/kg H~O; (8) 3% glutaraldehyde, 2% formaldehyde, 1% acrolein, and 2.5% dimethylsulphoxide
FIXATION OF DEVELOPING PROXIMAL TUBULES (DMSO) in 0.1 M sodium cacodylate buffer, pH 7.2 (21), 1750 mOsm/kg H~O. All solutions were freshly prepared and used at 20°C. Fixation by vascular perfusion. The rats were fastened with scotch tape on a wooden operating table which had a central round excavation to accommodate their backs. The abdomen was opened by a midline incision which continued beneath the left costal arch and the anterior thoracic wall cut away. The heart was then grasped with a pair of fine forceps and a fine needle (outer diameter 0.6 mm, 23 gauge) inserted into the left ventricle. The right auricle was finally cut open and the perfusion started immediately. During the first 15 sec of the perfusion the perfusate consisted either of 0.4% lidocain chloride (4) and 2% sucrose in 0.1 M sodium cacodylate buffer (pH 7.2; total osmolality 292 mOsm/kg H20) or a Tyrode solution (44) containing 0.4% lidocain chloride (pH 7.2; total osmolality 298 mOsm/kg H20. The rinse was followed by the fixative without interruption of the flow. Rinsing and fixative solutions were kept in separate bottles which were connected via a Y-junction to the perfusion tube. Dripping chambers were used for the control of flow. Adequate flow of perfusate was obtained by pressurising the bottles with compressed air to 100 mm Hg. The perfusion was continued for 3 min and the tissue then excised and immersed in the same fixative for another 2 hr. The perfusion technique was applied to all fixatives except osmium tetroxide. Fixation by dripping and immersion. Fixation by dripping the fixative on the surfaces of kidneys in vivo with intact vascular supply was performed essentially as described earlier (39, 4I). Only osmium tetroxide was applied in this way. After fixation for 15 min in situ small blocks were cut out and immersed in osmium tetroxide for one additional hour. Tissue fixed by immersion only was excised from kidneys of decapitated rats and immersed in the fixative for 2 hr. Primary immersion fixation was carried out with all fixatives. Postfixation, dehydration and embedding. Tissues fixed in aldehydes were rinsed in 0.1 M sodium cacodylate buffer, pH 7.2, and postfixed at 4°C in 1% osmium tetroxide in 0.029 M Veronal acetate buffer for 45 min. The tissues were then rinsed in Veronal acetate buffer and in some experiments treated with 0.5% uranyl acetate in Veronal acetate buffer, pH 5.0 for 45 min at 20°C (25) before dehydration. Tissue blocks from all experiments were dehydrated in increasing concentrations of acetone and embedded in Vestopal W (45). In some experiments tissue blocks were also dehydrated in ethanol and embedded in Epon 812 (36). In a few experiments tissues were dehydrated first in ethanol and then rinsed in styrene (27) before dehydration and embedding in Vestopal W.
141
Light and electron microscopy. One micron thick sections were cut and stained with toluidine blue (3) for light microscopy and used to determine the distance of any given tubule from the surface of the tissue block. In some cases serial thick sections were cut to identify the developmental stage of a tubule (28). Ultrathin sections were cut on an LKB Ultrotome III and stained with uranyl acetate (50) for 30-45 rain at 60°C (6) and/or lead citrate (43). The sections were examined at an accelerating voltage of 80 kV in a JEM 100 B electron microscope or a Siemens Elmiskop 1A using 200 #m condensor apertures and 60 #m and 50 ttm objective apertures in the respecrive microscopes. RESULTS
Macroscopic Observations on Tissue Fixed by Perfusion In animals perfused with glutaraldehyde the kidneys became hard and brown-yellow while those perfused with formaidehyde became light grey. When lidocain chloride was not included in the rinse it was usually impossible to satisfactorily perfuse the fixative.
Comparison Between Dif[erent Fixation Methods Electron microscopy revealed that the outstanding feature of kidneys fixed by vascular perfusion was a uniform preservation throughout the tissue (Figs. 1 and 2). In contrast kidneys fixed by dripping of fixative were preserved well only in the subcapsular zone of a tissue block containing renal vesicles or stage I (Fig. 3) and S-shaped bodies or stage II: The thickness of this zone was approximately 100 ttm. In tissue initially fixed by immersion, only the subcellular components of the cells in the peripherial zone of a tissue block were well preserved (compare Figs. 4 and 5). This zone was approximately 100 #m thick in tissue fixed with glutaraldehyde and osmium tetroxide and about 200 ttm with formaldehyde-containing fixatives. The lumens in renal vesicles and S-shaped bodies were open with all fixation methods. However, the lumens of proximal tubules in late developmental stages (stage
FIG. 1. Survey picture of a renal vesicle (stage I) fixed by vascular perfusion with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer. The renal vesicle is located just below the renal capsule (RC). The cells are radially oriented from the lumen (LU) and the cytoplasm of the cells have a uniform electron density. Collecting tubule (CT). The tissue was treated with uranyl acetate e n bloc and embedded in Vestopal W. Section stained with lead citrate. × 2100. F~G. 2. Survey picture of proximal tubules in stage IV fixed and prepared as in Fig. 1. The tubules are located approximately 200 ~m from the kidney surface. The brush border is constant in width and the cytoplasm of the cells have a uniform electron density. A few wide intercellular spaces are seen (arrows). Section stained with uranyl acetate and lead citrate. × 1600. 142
Fins. 3-5. Different developmental stages fixed with 1% osmium tetroxide in Veronal acetate buffer. Tissue treated with uranyl acetate e n bloc and embedded in Vestopal W. F~a. 3. Survey picture of a renal vesicle within 50 ~m of the kidney surface fixed by dripping. The cytoplasm of the cells have a uniform electron density. Section stained with lead citrate, x 3 600. Fro. 4. Part of an S-shaped body within 50 t~m from the kidney surface fixed by immersion. The cells exhibit 143
LARS LARSSON
144
TABLE I EFFECTS OF ~IFFERENT FIXATIVES ON THE ULTRASTRUCTURE OF RENAL VESICLE AND S-SHAPED BODY
Fixativesa
1. 6% glutaraldehyde 0.1 M sodium cacodylate 2, 6% glutaraldehyde 0.01 M sodium cacodylate 3. 3% glutaraldehyde 0.1 M sodium cacodylate 4. 1% osmium tetroxide Veronal acetate made isotonic with salts 5. 4% formaldehyde 0.1 M sodium cacodylate 6. 5% glutaraldehyde 4% formaldehyde 0.1 M sodium cacodylate 7. 2.5% glutaraldehyde 2% formaldehyde 8. 3% glutaraldehyde 2% formaldehyde 1% acrolein 0.1 M sodium cacodylate
Material in lumen
Cell membrane
Width of intercellular spaces
Electron density ofthe cytoplasm
absent
intact
sometimes
broken
narrow, some wide narrow or absent
often
broken
narrow
medium
absent
intact
narrow
medium
absent
intact
variable
medium
absent
intact
wide
high
absent
intact
wide
medium
absent
intact
wide
high
medium low
a Fixative 1-3 and 5-8 applied by perfusion; fixative 4 applied by immersion or dripping. I I I a n d s t a g e IV) were c o l l a p s e d in t i s s u e f i x e d b y i m m e r s i o n a n d in t i s s u e f i x e d b y d r i p p i n g w h i l e t h e y w e r e f u l l y o p e n in t i s s u e f i x e d b y v a s c u l a r p e r f u s i o n (Fig. 2 a n d a l s o F i g . 12 in ref. 28). W h e n f o r m a l d e h y d e - c o n t a i n i n g f i x a t i v e s were a p p l i e d b y i m m e r s i o n t h e i n t e r c e l l u l a r s p a c e s were larger than when the same fixatives were perfused. T h e u l t r a s t r u c t u r e of cells a n d 2 u b c e l l u l a r c o m p o n e n t s in t h e r e n a l v e s i c l e a n d t h e S - s h a p e d b o d y w a s e s s e n t i a l l y t h e s a m e in t i s s u e f i x e d in o s m i u m t e t r o x i d e b o t h w h e n t h e f i x a t i v e was a p p l i e d b y i m m e r s i o n or d r i p p i n g . P r o x i m a l t u b u l e s in s t a g e III a n d s t a g e IV w h i c h a r e l o c a t e d m o r e t h a n 100 u m f r o m t h e s u r f a c e of t h e k i d n e y w e r e
always poorly fixed by dripping. I m m e r s i o n in e i t h e r o s m i u m or g l u t a r a l d e h y d e p r e s e r v e d subcellular c o m p o n e n t s e q u a l l y well in all d e v e l o p m e n t a l s t a g e s in t h e s u p e r f i c i a l l a y e r s of t h e t i s s u e b l o c k . I n t i s s u e p r i m a r i l y f i x e d b y i m m e r s i o n in formaldehyde-containing fixatives the subc e l l u l a r c o m p o n e n t s in t h e s u p e r f i c i a l l a y ers of t i s s u e b l o c k s s h o w e d t h e s a m e p r e s e r v a t i o n as in t i s s u e f i x e d b y p e r f u s i o n in t h e same fixatives.
Effects of Different Fixatives Glutaraldehyde. T h e c o n c e n t r a t i o n
of the fixative greatly influenced the appeara n c e of t h e t i s s u e ( T a b l e I). A f t e r p e r f u s i o n f i x a t i o n w i t h 6% g l u t a r a l d e h y d e in 0.1 M
a uniform cytoplasmic electron density and are well preserved. Note the smooth luminal cell membrane. Section stained with uranyl acetate, lead citrate. × 10 000. FIG. 5. Part of an S-shaped body located approximately 150 um from the kidney surface. The luminal cell membrane is uneven and apical parts of the cytoplasm appear swollen and have protruded into the lumen (LU). Some mitochondria are swollen and have an uneven electron density (arrows). Section stained with uranyl acetate and lead citrate. × 7100.
FIXATION OF DEVELOPING PROXIMAL TUBULES sodium cacodylate buffer the ground substance in the cytoplasm of the cells showed a uniform electron density, the mitochondria had a homogenous m a t r i x and cell m e m b r a n e s and c y t o m e m b r a n e s of the Golgi a p p a r a t u s and endoplasmic reticulure were intact. These characteristics of well preserved tissue were found in tubule cells of all d e v e l o p m e n t a l stages throughout the renal cortex (Figs. 1, 2, and 8, see also Figs. 11 and 12 in ref. 28). However, when the fixative concentration was lowered to 3% in the same buffer renal vesicles and S-shaped bodies usually showed features typical of poorly preserved tissue. Thus, apical cell m e m b r a n e s often appeared disrupted and apical parts of the cytoplasm p r o t r u d e d into the lumen (Fig. 6). T h e lateral cell m e m b r a n e s were also
145
frequently broken. Proximal tubules in stages III and IV showed no obvious differences with respect to the quality of fixation following perfusion with 3% or 6% glutaraldehyde. The fixative vehicle was of particular importance for the shape of the cells. Cells in the renal vesicles and S-shaped bodies fixed in 6% glutaraldehyde in 0.01 sodium cacodylate were swollen as indicated by a very e l e c t r o n t r a n s l u c e n t c y t o p l a s m i c ground substance (Fig. 7). The swelling was most pronounced in the apical parts of the cells. T h e matrix of the mitochondria was not homogenous (Fig. 7). T h e cell m e m b r a n e s were often disrupted and cytoplasmic components sometimes located in the tubule lumen. Proximal tubules in stages III and IV showed the same general
Fro. 6. Apical part of a stage I tubule fixed with 3% glutaraldebyde in 0.1 M sodium cacodylate buffer. Note the disrupted apical cell membrane and cytoplasmic components in the tubule lumen (LU). Mitochondria (M), Golgi apparatus (G) and rough surfaced endoplasmic reticulum (RER) appear well preserved. Tissue embedded in Epon. Section stained with uranyl acetate and lead citrate. × 17 600.
146
LARS LARSSON
changes b u t were affected to a lesser degree. T h e intercellular spaces in the proximal tubule of all developmental stages were different in tissue fixed with 6% glutaraldehyde in 0.1 M than in 0.01 M sodium cacodylate buffer (Table I). Occasional wide intercellular spaces were found with 0.1 M but not with 0.01 M sodium cacodylate. Lateral plasma m e m b r a n e s in proximal tubules of stage IV were often apposed giving rise to five-layered m e m b r a n e complexes. Such complexes were longer in tissue fixed with glutaraldehyde in 0.01 M sodium cacodylate (Fig. 9) than in tissue fixed with glutaraldehyde in 0.1 M sodium cacodylate (Fig. 10). These findings were i n d e p e n d e n t of the postfixation and dehydration methods.
Formaldehyde and Combined Fixatives All formaldehyde-containing fixatives caused an a p p a r e n t shrinkage of the cells when c o m p a r e d with glutaraldehyde-fixed tissue as indicated by large intercellular spaces and an electron dense cytoplasm. This a p p a r e n t shrinkage was more pronounced in early than in late developm e n t a l stages of the proximal tubules (Figs. 11-13). F o r m a l d e h y d e alone at a concentration of 4% produced scattered large intercellular spaces in renal vesicles and S-shaped bodies (Table I) b u t only occasionally in proximal tubules at stages III and IV. T h e other formaldehyde containing fixatives produced large intercellular spaces in tubules just b e n e a t h the renal capsule b u t the spaces gradually decreased in width toward the interior of the cortex (Fig. 11) regardless of the fixation method. M e m b r a n e l i k e fragments and myelinlike
figures were sometimes seen in the intercellular spaces, b u t lateral and apical cell m e m b r a n e s were usually intact. T h e cytoplasm of cells fixed by perfusion with 4% formaldehyde combined with 5% glutaraldehyde (Figs. 11-13) or with 3% glutaraldehyde combined with 2% formaldehyde, 1% acrolein, and 2.5% D M S O was more dense t h a n with all the other fixatives. T h e r e was no obvious evidence t h a t the latter fixative combination p e n e t r a t e d and preserved the interior of excised tissue blocks better t h a n the other aldehyde mixtures of fixatives. DISCUSSION T h e present comparison of different fixation m e t h o d s demonstrates t h a t vascular perfusion with glutaraldehyde provides uniform fixation throughout the developing renal cortex. All other methods of applying the fixative caused variable qualities of fixation at different distances from the tissue surface. These observations are consistent with results obtained by the application of perfusion methods to other developing tissues (i, 22, 34). In the case of the kidney the uniform preservation of the developing cortex is significant since only under such conditions is it possible to m a k e a meaningful comparison between different developmental stages of the proximal tubules located at different levels below the kidney surface. Perfusion fixation should also be advantageous in histochemical studies since any diffusion of enzymes will be less by this m e t h o d t h a n in immersion fixation procedures where the fixative penetrates slowly (17). T h e present study demonstrates t h a t the
Fro. 7. Apical parts of a stage I tubule fixed with 6% glutaraldehyde in 0.01 M sodium cacodylate buffer. The cytoplasm is apparently swollen and many areas appear empty (arrows). Most mitochondria have a low and uneven electron density. Compare with Fig. 8 where the tissue is fixed in 6% glutaraldehyde but in 0.1 M sodium cacodylate buffer. The tissue was treated with uranyl acetate en bloc and embedded in Vestopal W. Sections stained with uranyl acetate and lead citrate. × 17 100. Fro. 8. Corresponding area as in Fig. 7 but the tissue is fixed with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer. The cytoplasm and mitochondria have a uniform electron density without evidence of swelling Embedding and staining as in Fig. 7. × 17 100.
FIXATION OF DEVELOPING PROXIMAL TUBULES
147
148
LARS LARSSON
FIG. 9. Basal parts of a proximal tubule in stage IV fixed with 6% glutaraldehyde in 0.01 M sodium cacodylate buffer. Note that the intercellular spaces are almost absent and that the cell membranes have formed long five-layered membrane complexes (arrows). Compare with Fig. 10 where the tissue is fixed with 6% glutaraldehyde but in 0.1 M sodium cacodylate buffer. The tissue was treated with uranyl acetate en bloc and embedded in Vestopal W. Sections stained with uranyl acetate and lead citrate. × 81 000. FIG. 10. Corresponding area as in Fig. 9 but the tissue is fixed with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer. Irregular intercellular spaces (IS) are seen and adjacent cell membranes are occasionally apposed forming five-layered membrane complexes (arrows). Embedding and staining as in Fig. 9. × 81 000. p e r f u s i o n f i x a t i o n m e t h o d w h i c h is s i m i l a r to t h a t e m p l o y e d for a d u l t r e n a l t u b u l e s (37) is also u s e f u l for d e v e l o p i n g r e n a l t u b u l e s p r o v i d e d t h a t t h e c o m p o s i t i o n of t h e f i x a t i v e is m o d i f i e d . T h e m o s t i m p o r tant modification concerns the concentrat i o n of g l u t a r a l d e h y d e w h i c h m u s t be r a i s e d to 6% in o r d e r to g e t s a t i s f a c t o r y
p r e s e r v a t i o n of t h e p l a s m a m e m b r a n e s in t h e first t w o d e v e l o p m e n t a l s t a g e s . In cont r a s t good f i x a t i o n of a d u l t p r o x i m a l tub u l e s is o b t a i n e d w i t h a c o n c e n t r a t i o n of g l u t a r a l d e h y d e of 1% or e v e n less (38). T h e r e q u i r e m e n t for a h i g h g l u t a r a l d e h y d e conc e n t r a t i o n m a y d e r i v e f r o m t h e p r e s e n c e in t h e d e v e l o p i n g r e n a l c o r t e x of a less r i c h
FIGS. 11-13. Different stages fixed by immersion with 4% formaldehyde and 5% glutaraldehyde in 0.1 M sodium cacodylate buffer and embedded in Vestopal. Sections stained with uranyl acetate and lead citrate. FIa. 11. Large intercellular spaces are seen in a renal vesicle or stage I (I) and a collecting tubule (CT) just below the renal capsule (RC). The intercellular spaces are less pronounced in stage III (III) deeper in the cortex. The cytoplasm is very electron dense in all cells. × 1700. FIa. 12. Part of stage I tubule. There are large intercellular spaces (IS) and the cytoplasm is very electron dense. It is difficult to identify the mitochondria (arrows). × 12 600. Fro. 13. Part of stage IV tubule. The intercellular spaces have approximately the same width as in tissue fixed with 6% glutaraldehyde in 0.1 M sodium cacodylate (compare with Fig. 10) but the mitochondria (M) are difficult to identify. × 27 900.
149
150
LARS LARSSON
vascular supply in the outer cortex t h a n in the inner cortex (35). In contrast in the adult kidney the vascular supply is rich t h r o u g h o u t cortex (14). Since glutaraldehyde has been shown to p e n e t r a t e faster at higher concentrations (18) the high concentration employed in this study m a y be efficient to overcome the disadvantage of the less extensive vasculature of the developing kidney. Recent studies have shown t h a t there m a y be an intercellular p a t h w a y of small molecules from one side of an epithelium to the other (11, 16). In any a t t e m p t to gain structural evidence for this p a t h w a y it is clearly i m p o r t a n t to ensure in the first instance t h a t the tissue is well preserved. This is e m p h a s i z e d by the present observation t h a t adjacent cell m e m b r a n e s were more frequently apposed in 0.01 M buffer t h a n in 0.1 M buffer. This shows t h a t the composition of the vehicle of the fixative influences the width of the intercellular spaces in developing as in adult renal tubules (38). It is therefore necessary to consider possible fixation alteration of the tissue before unequivocal conclusions can be drawn with regard to the a p p e a r a n c e of the intercellular spaces. T h e frequency and length of the basal five-layered m e m b r a n e complexes is dep e n d e n t upon the osmolality of the fixative since almost all these complexes disappeared when the tissue was fixed with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer. It is likely t h a t more m e m b r a n e complexes are being formed by the fixatives with a hypoosmolal buffer. It is unlikely t h a t they are true cell contacts since in other tissues these resist large changes in the osmolality of the vehicle of the fixative (5). Small variations in the composition of the vehicle of the fixative (0.07 M-0.1 M sodium cacodylate) have no obvious effects on the width of the intercellular spaces (28).
Tissue fixed with formaldehyde-containing fixatives showed, i n d e p e n d e n t of the
m e t h o d of application, wider intercellular spaces in early t h a n in late developmental stages. This m a y indicate t h a t early develo p m e n t a l stages are more sensitive to osmotic variations in the fixative t h a n the later stages in newborn rat kidney. Adult proximal tubule cells also seem to be more sensitive to the osmolality of the fixative t h a n proximal tubule cells of stages III and IV since vascular perfusion with 6% glutaraldehyde in 0.1 M sodium cacodylate showed very wide intercellular spaces in all proximal tubules t h r o u g h o u t the renal cortex in adult animals (30). These observations show t h a t a tissue at different develo p m e n t a l stages m a y react differently to a certain fixative and indicate the necessity to explore the effects of a fixative at each developmental stage under investigation. REFERENCES 1. ABRUNHOSA,R., J. Ultrastruct. Res. 41,176 (1972). 2. AOKI,A., Anat. Rec. 155, 339 (1966). 3. BENCOSME, S., STONE, R. S., LATTA, It., AND MADDEN, S. C., J. Biophys. Biochem. Cytol. 5,
508 (1959). 4. BOHMAN, S.-O., J. Ultrastruct. Res. 47, 329
(1974). 5. BRIGHTMAN,M. W., ANDREESE, W. S., J. Cell Biol.
40, 648 (1969). 6. BRODY, I., J. Ultrastruct. Res. 2, 482 (1959). 7. CALEY, D. W., AND MAXWELL, D. S., J. Comp. Neurol. 138, 31 (1970). 8. CLARK, S. L., g. Biophys. Biochem. Cytol. 3, 349
(1957). 9. COHEN,A. I., Develop. Biol. 3, 297 (1961). 10. DEHAAN,R. L., in DeHaan, R. L. and Ursprung, H. (Eds.), Organogenesis, p. 377. Holt, Rinehart and Winston, New York, 1965. 11. DmONA,D. R., Nature New Biol. 238, 179 (1972). 12. DUNCAN, D., Texas Rep. Biol. Med. 15, 367 (1957). 13. FAHIMI, H. D., AND DROCHMANS,P., J. Microsc. (Paris) 4, 725 (1965). 14. FOURMAN, J., AND MOFFAT, D. B., The blood
vessels of the kidney. Blackwell, Oxford, 1971. 15. FELDMAN,J. S., VAZQUEZ,J. J., ANDKURTZ, S. M., J. Biophys. Biochem. Cytol. ll, 365 (1961). 16. GIEBISCH,G., BOULPEAP,E. L., ANDWHITTEMBURY, G., Phil. Trans. Roy. Soc. Lond. B. 262, 175
(1971). 17. GOLDFISCHER,S., ESSNER, E., ANDNOVIKOFF,A. B., J. Histochern. Cytochem. 12, 72 (1964).
FIXATION OF DEVELOPING PROXIMAL TUBULES 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
32. 33. 34.
35. 36. 37. 38.
151
HOPWOOD,D., Histochem. J. 1, 323 (1969). (1966). HUBER, G. C., Am. J. Anat. 4, Suppl, 1 (1905). 39. MAUNSEACH,A. B., MADDEN,S. C., ANDLATTA,H., JOKELAINEN,P., Acta Anat. 52, Suppl, 47 (1963). J. Ultrastruct. Res. 6, 511 (1962). KALT, M. R., AND TANDLER, B., J. Ultrastruct. 40. NILSSON, S. E. G., J. Ultrastruct. Res. ll, 581 Res. 36, 633 (1971). (1964). KARLSSON,U., J. Ultrastruct. Res. 17, 158 (1967). 41. PEASE, D. C., Anat. Rec. 121, 701 (1955). KARNOVSKY,M. J., J. Cell Biol. 27, 137 A (1965). 42. PEASE, D. C., ibid. 142, 342 (1962). KAZIEMIERCZAK, J., Acta Pathol. Microbiol. 43. REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). Scand. Suppl. 218 (1971). 44. ROMEIS, B., Mikroskopische Technik, p. 31. O1KELLENBERGER,E., RYTER, A., ANDSI~CHAUD,J., J. denburg, Mtinchen, Wien, 1968. Biophys. Biochem. Cytol. 4, 671 (1958). 45. RYTER, A., ANDKELLENBERGER,E., J. Ultrastruct. KURTZ, S. M., Exp. Cell Res. 14, 355 (1958). Res. 2, 200 (1958). KURTZ, S. M., J. Ultrastruct. Res. 5, 468 (1961). 46. SABATINI,D. D., BENSCH,K., ANDBARRNETT,R. J., LARSSON,L., J. Ultrastruct. Res. 51, 119 (1975). J. Cell Biol. 17, 19 (i963). LARSSON,L., to be published. 47. SJI~STRAND,f. S., Electron Microscopy of Cells LARSSON,L., unpublished observations. and Tissues, Vol l, p. 144. Academic Press, New LARSSON, L., MAUNSEACH,A. B., SAXI~N, L., AND York, 1967. WARTIOVAARA,J., J. Ultrastruct. Res. 29, 570 48. SuzuKI, Y., J. Electron microsc. 6, 52 (1958). (1969). 49. WARTIOVAARA,J., Ann. Med. Exp. Biol. Fenn. 44, LARSSON,L., AND MAUI~SBACH,A. B., to be pub4O9 (1966). lished. 50. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, LEESON, T. S., Amer. J. Anat. 105, 165 (1959). 475 (1958). LESKES, A., SIEKEVITZ, P., AND PALADE, G. E., J. 51. WEBEER,W. A., ANDBLACKBOURN,J., Lab. Invest. Cell Biol. 49, 264 (1971). 23, 1 (1970). LJUNGQVIST,A., Acta Paediat. 52, 443 (1963). 52. VERNIER, R. L., AND BIRCH-ANDERSEN, A., J. LUFT, J., J. Biophys. Biochem. Cytol. 9, 409 Pediat. 60, 754 (1962). (1961). 53. YOSHIMURA,F., ANDNAKAMURA,M., Ohajimas Fol. MAUNSBACH,A. B., J. Ultrastruct. Res. 15, 242 Anat. Jap. 41, 121 (1965). (1966). 54. ZAMEONI,L., AND DE MARTINO, C., J. Ultrastruct. MAUNSBACH,A. B., J. Ultrastruct. Res. 15, 283 Res. 16, 399 (1966).
Effects of Different Fixatives on the Ultrastructure of the Developing Proximal Tubule in the Rat Kidney LARS LARSSON
Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark Received August 13, 1974; accepted September 23, 1974 The fine structure of developing renal tubule cells was evaluated following fixation by different fixatives (osmium tetroxide, glutaraldehyde, formaldehyde, and different combinations of aldehydes) appIied either by vascular perfusion, immersion or dripping on the kidney surface. Vascular perfusion with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer gave a uniform preservation of the developing renal cortex and facilitated a detailed comparison between different developmental stages of the proximal tubule in the same animal.
MATERIALS AND METHODS
INTRODUCTION
Embryonic tissues are generally considered to be difficult to fix for electron microscopy (7, 9, 10, 12, I5, 40) and in the case of renal tissue (8) particular difficulty has been encountered in obtaining uniform preservation throughout the cortex which contains different developmental stages of the nephron (19). In previous studies of developing kidney tubules, the tissue has been fixed by immersion (2, 8, 20, 24, 26, 31, 33, 48, 49, 51-54). However, as pointed out by Clark (8), this procedure results in fixation of different quality at different levels of the renal cortex and makes it very difficult to analyze the fine structure of tubules of different developmental stages. This paper reports results obtained with various fixatives applied in different ways and evaluates the results with particular emphasis on the developing proximal tubule. The conclusions from this work have served as the basis for a series ofultrastructural studies of the developing proximal tubule (28, 29, 32). The definition of the developmental stages of the proximal tubule is the same as presented in detail in the adjoining paper
(28). 140 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
Fifty-six male Wistar rats from 4 to 48 hours old were used. Each experimental group consisted of three to seven rats. The rats were anaesthetized by intraperitoneal injections of sodium pentobarbital (Nembutal, Abbotts Laboratories) of 50 mg/kg body wt. Composition of fixatives. Glutaraldehyde (25%, Merck) was purified by shaking with charcoal (13). The concentration of the glutaraldehyde was determined by measuring the freezing point depression of the solution with an Advanced Osmometer (Model 3L) and taking 1% glutaraldehyde to have an osmolality of 100 mOsm/kg H20 (13). Formaldehyde solutions were prepared as described by Pease (42). Each of the following fixatives was used on one group of animals: (1) 6% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, 828 mOsm/kg H20; (2) 6% glutaraldehyde in 0.01 M sodium cacodylate buffer, pH 7.2, 616 mOsm/kg H20; (3) 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, 506 mOsm/kg H20; (4) 1% osmium tetroxide in 0.029 M Veronal acetate buffer made isotonic with salts (47), pH 7.2, 349 mOsm/kg H20; (5) 4% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, 1450 mOsm/kg H20; (6) 5% glutaraldehyde and 4% formaldehyde in 0.1 M sodium cacodylate buffer (23), pH 7.2, 1990 mOsm/kg H20; (7) 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer (23), pH 7.2, 1080 mOsm/kg H~O; (8) 3% glutaraldehyde, 2% formaldehyde, 1% acrolein, and 2.5% dimethylsulphoxide
FIXATION OF DEVELOPING PROXIMAL TUBULES (DMSO) in 0.1 M sodium cacodylate buffer, pH 7.2 (21), 1750 mOsm/kg H~O. All solutions were freshly prepared and used at 20°C. Fixation by vascular perfusion. The rats were fastened with scotch tape on a wooden operating table which had a central round excavation to accommodate their backs. The abdomen was opened by a midline incision which continued beneath the left costal arch and the anterior thoracic wall cut away. The heart was then grasped with a pair of fine forceps and a fine needle (outer diameter 0.6 mm, 23 gauge) inserted into the left ventricle. The right auricle was finally cut open and the perfusion started immediately. During the first 15 sec of the perfusion the perfusate consisted either of 0.4% lidocain chloride (4) and 2% sucrose in 0.1 M sodium cacodylate buffer (pH 7.2; total osmolality 292 mOsm/kg H20) or a Tyrode solution (44) containing 0.4% lidocain chloride (pH 7.2; total osmolality 298 mOsm/kg H20. The rinse was followed by the fixative without interruption of the flow. Rinsing and fixative solutions were kept in separate bottles which were connected via a Y-junction to the perfusion tube. Dripping chambers were used for the control of flow. Adequate flow of perfusate was obtained by pressurising the bottles with compressed air to 100 mm Hg. The perfusion was continued for 3 min and the tissue then excised and immersed in the same fixative for another 2 hr. The perfusion technique was applied to all fixatives except osmium tetroxide. Fixation by dripping and immersion. Fixation by dripping the fixative on the surfaces of kidneys in vivo with intact vascular supply was performed essentially as described earlier (39, 4I). Only osmium tetroxide was applied in this way. After fixation for 15 min in situ small blocks were cut out and immersed in osmium tetroxide for one additional hour. Tissue fixed by immersion only was excised from kidneys of decapitated rats and immersed in the fixative for 2 hr. Primary immersion fixation was carried out with all fixatives. Postfixation, dehydration and embedding. Tissues fixed in aldehydes were rinsed in 0.1 M sodium cacodylate buffer, pH 7.2, and postfixed at 4°C in 1% osmium tetroxide in 0.029 M Veronal acetate buffer for 45 min. The tissues were then rinsed in Veronal acetate buffer and in some experiments treated with 0.5% uranyl acetate in Veronal acetate buffer, pH 5.0 for 45 min at 20°C (25) before dehydration. Tissue blocks from all experiments were dehydrated in increasing concentrations of acetone and embedded in Vestopal W (45). In some experiments tissue blocks were also dehydrated in ethanol and embedded in Epon 812 (36). In a few experiments tissues were dehydrated first in ethanol and then rinsed in styrene (27) before dehydration and embedding in Vestopal W.
141
Light and electron microscopy. One micron thick sections were cut and stained with toluidine blue (3) for light microscopy and used to determine the distance of any given tubule from the surface of the tissue block. In some cases serial thick sections were cut to identify the developmental stage of a tubule (28). Ultrathin sections were cut on an LKB Ultrotome III and stained with uranyl acetate (50) for 30-45 rain at 60°C (6) and/or lead citrate (43). The sections were examined at an accelerating voltage of 80 kV in a JEM 100 B electron microscope or a Siemens Elmiskop 1A using 200 #m condensor apertures and 60 #m and 50 ttm objective apertures in the respecrive microscopes. RESULTS
Macroscopic Observations on Tissue Fixed by Perfusion In animals perfused with glutaraldehyde the kidneys became hard and brown-yellow while those perfused with formaidehyde became light grey. When lidocain chloride was not included in the rinse it was usually impossible to satisfactorily perfuse the fixative.
Comparison Between Dif[erent Fixation Methods Electron microscopy revealed that the outstanding feature of kidneys fixed by vascular perfusion was a uniform preservation throughout the tissue (Figs. 1 and 2). In contrast kidneys fixed by dripping of fixative were preserved well only in the subcapsular zone of a tissue block containing renal vesicles or stage I (Fig. 3) and S-shaped bodies or stage II: The thickness of this zone was approximately 100 ttm. In tissue initially fixed by immersion, only the subcellular components of the cells in the peripherial zone of a tissue block were well preserved (compare Figs. 4 and 5). This zone was approximately 100 #m thick in tissue fixed with glutaraldehyde and osmium tetroxide and about 200 ttm with formaldehyde-containing fixatives. The lumens in renal vesicles and S-shaped bodies were open with all fixation methods. However, the lumens of proximal tubules in late developmental stages (stage
FIG. 1. Survey picture of a renal vesicle (stage I) fixed by vascular perfusion with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer. The renal vesicle is located just below the renal capsule (RC). The cells are radially oriented from the lumen (LU) and the cytoplasm of the cells have a uniform electron density. Collecting tubule (CT). The tissue was treated with uranyl acetate e n bloc and embedded in Vestopal W. Section stained with lead citrate. × 2100. F~G. 2. Survey picture of proximal tubules in stage IV fixed and prepared as in Fig. 1. The tubules are located approximately 200 ~m from the kidney surface. The brush border is constant in width and the cytoplasm of the cells have a uniform electron density. A few wide intercellular spaces are seen (arrows). Section stained with uranyl acetate and lead citrate. × 1600. 142
Fins. 3-5. Different developmental stages fixed with 1% osmium tetroxide in Veronal acetate buffer. Tissue treated with uranyl acetate e n bloc and embedded in Vestopal W. F~a. 3. Survey picture of a renal vesicle within 50 ~m of the kidney surface fixed by dripping. The cytoplasm of the cells have a uniform electron density. Section stained with lead citrate, x 3 600. Fro. 4. Part of an S-shaped body within 50 t~m from the kidney surface fixed by immersion. The cells exhibit 143
LARS LARSSON
144
TABLE I EFFECTS OF ~IFFERENT FIXATIVES ON THE ULTRASTRUCTURE OF RENAL VESICLE AND S-SHAPED BODY
Fixativesa
1. 6% glutaraldehyde 0.1 M sodium cacodylate 2, 6% glutaraldehyde 0.01 M sodium cacodylate 3. 3% glutaraldehyde 0.1 M sodium cacodylate 4. 1% osmium tetroxide Veronal acetate made isotonic with salts 5. 4% formaldehyde 0.1 M sodium cacodylate 6. 5% glutaraldehyde 4% formaldehyde 0.1 M sodium cacodylate 7. 2.5% glutaraldehyde 2% formaldehyde 8. 3% glutaraldehyde 2% formaldehyde 1% acrolein 0.1 M sodium cacodylate
Material in lumen
Cell membrane
Width of intercellular spaces
Electron density ofthe cytoplasm
absent
intact
sometimes
broken
narrow, some wide narrow or absent
often
broken
narrow
medium
absent
intact
narrow
medium
absent
intact
variable
medium
absent
intact
wide
high
absent
intact
wide
medium
absent
intact
wide
high
medium low
a Fixative 1-3 and 5-8 applied by perfusion; fixative 4 applied by immersion or dripping. I I I a n d s t a g e IV) were c o l l a p s e d in t i s s u e f i x e d b y i m m e r s i o n a n d in t i s s u e f i x e d b y d r i p p i n g w h i l e t h e y w e r e f u l l y o p e n in t i s s u e f i x e d b y v a s c u l a r p e r f u s i o n (Fig. 2 a n d a l s o F i g . 12 in ref. 28). W h e n f o r m a l d e h y d e - c o n t a i n i n g f i x a t i v e s were a p p l i e d b y i m m e r s i o n t h e i n t e r c e l l u l a r s p a c e s were larger than when the same fixatives were perfused. T h e u l t r a s t r u c t u r e of cells a n d 2 u b c e l l u l a r c o m p o n e n t s in t h e r e n a l v e s i c l e a n d t h e S - s h a p e d b o d y w a s e s s e n t i a l l y t h e s a m e in t i s s u e f i x e d in o s m i u m t e t r o x i d e b o t h w h e n t h e f i x a t i v e was a p p l i e d b y i m m e r s i o n or d r i p p i n g . P r o x i m a l t u b u l e s in s t a g e III a n d s t a g e IV w h i c h a r e l o c a t e d m o r e t h a n 100 u m f r o m t h e s u r f a c e of t h e k i d n e y w e r e
always poorly fixed by dripping. I m m e r s i o n in e i t h e r o s m i u m or g l u t a r a l d e h y d e p r e s e r v e d subcellular c o m p o n e n t s e q u a l l y well in all d e v e l o p m e n t a l s t a g e s in t h e s u p e r f i c i a l l a y e r s of t h e t i s s u e b l o c k . I n t i s s u e p r i m a r i l y f i x e d b y i m m e r s i o n in formaldehyde-containing fixatives the subc e l l u l a r c o m p o n e n t s in t h e s u p e r f i c i a l l a y ers of t i s s u e b l o c k s s h o w e d t h e s a m e p r e s e r v a t i o n as in t i s s u e f i x e d b y p e r f u s i o n in t h e same fixatives.
Effects of Different Fixatives Glutaraldehyde. T h e c o n c e n t r a t i o n
of the fixative greatly influenced the appeara n c e of t h e t i s s u e ( T a b l e I). A f t e r p e r f u s i o n f i x a t i o n w i t h 6% g l u t a r a l d e h y d e in 0.1 M
a uniform cytoplasmic electron density and are well preserved. Note the smooth luminal cell membrane. Section stained with uranyl acetate, lead citrate. × 10 000. FIG. 5. Part of an S-shaped body located approximately 150 um from the kidney surface. The luminal cell membrane is uneven and apical parts of the cytoplasm appear swollen and have protruded into the lumen (LU). Some mitochondria are swollen and have an uneven electron density (arrows). Section stained with uranyl acetate and lead citrate. × 7100.
FIXATION OF DEVELOPING PROXIMAL TUBULES sodium cacodylate buffer the ground substance in the cytoplasm of the cells showed a uniform electron density, the mitochondria had a homogenous m a t r i x and cell m e m b r a n e s and c y t o m e m b r a n e s of the Golgi a p p a r a t u s and endoplasmic reticulure were intact. These characteristics of well preserved tissue were found in tubule cells of all d e v e l o p m e n t a l stages throughout the renal cortex (Figs. 1, 2, and 8, see also Figs. 11 and 12 in ref. 28). However, when the fixative concentration was lowered to 3% in the same buffer renal vesicles and S-shaped bodies usually showed features typical of poorly preserved tissue. Thus, apical cell m e m b r a n e s often appeared disrupted and apical parts of the cytoplasm p r o t r u d e d into the lumen (Fig. 6). T h e lateral cell m e m b r a n e s were also
145
frequently broken. Proximal tubules in stages III and IV showed no obvious differences with respect to the quality of fixation following perfusion with 3% or 6% glutaraldehyde. The fixative vehicle was of particular importance for the shape of the cells. Cells in the renal vesicles and S-shaped bodies fixed in 6% glutaraldehyde in 0.01 sodium cacodylate were swollen as indicated by a very e l e c t r o n t r a n s l u c e n t c y t o p l a s m i c ground substance (Fig. 7). The swelling was most pronounced in the apical parts of the cells. T h e matrix of the mitochondria was not homogenous (Fig. 7). T h e cell m e m b r a n e s were often disrupted and cytoplasmic components sometimes located in the tubule lumen. Proximal tubules in stages III and IV showed the same general
Fro. 6. Apical part of a stage I tubule fixed with 3% glutaraldebyde in 0.1 M sodium cacodylate buffer. Note the disrupted apical cell membrane and cytoplasmic components in the tubule lumen (LU). Mitochondria (M), Golgi apparatus (G) and rough surfaced endoplasmic reticulum (RER) appear well preserved. Tissue embedded in Epon. Section stained with uranyl acetate and lead citrate. × 17 600.
146
LARS LARSSON
changes b u t were affected to a lesser degree. T h e intercellular spaces in the proximal tubule of all developmental stages were different in tissue fixed with 6% glutaraldehyde in 0.1 M than in 0.01 M sodium cacodylate buffer (Table I). Occasional wide intercellular spaces were found with 0.1 M but not with 0.01 M sodium cacodylate. Lateral plasma m e m b r a n e s in proximal tubules of stage IV were often apposed giving rise to five-layered m e m b r a n e complexes. Such complexes were longer in tissue fixed with glutaraldehyde in 0.01 M sodium cacodylate (Fig. 9) than in tissue fixed with glutaraldehyde in 0.1 M sodium cacodylate (Fig. 10). These findings were i n d e p e n d e n t of the postfixation and dehydration methods.
Formaldehyde and Combined Fixatives All formaldehyde-containing fixatives caused an a p p a r e n t shrinkage of the cells when c o m p a r e d with glutaraldehyde-fixed tissue as indicated by large intercellular spaces and an electron dense cytoplasm. This a p p a r e n t shrinkage was more pronounced in early than in late developm e n t a l stages of the proximal tubules (Figs. 11-13). F o r m a l d e h y d e alone at a concentration of 4% produced scattered large intercellular spaces in renal vesicles and S-shaped bodies (Table I) b u t only occasionally in proximal tubules at stages III and IV. T h e other formaldehyde containing fixatives produced large intercellular spaces in tubules just b e n e a t h the renal capsule b u t the spaces gradually decreased in width toward the interior of the cortex (Fig. 11) regardless of the fixation method. M e m b r a n e l i k e fragments and myelinlike
figures were sometimes seen in the intercellular spaces, b u t lateral and apical cell m e m b r a n e s were usually intact. T h e cytoplasm of cells fixed by perfusion with 4% formaldehyde combined with 5% glutaraldehyde (Figs. 11-13) or with 3% glutaraldehyde combined with 2% formaldehyde, 1% acrolein, and 2.5% D M S O was more dense t h a n with all the other fixatives. T h e r e was no obvious evidence t h a t the latter fixative combination p e n e t r a t e d and preserved the interior of excised tissue blocks better t h a n the other aldehyde mixtures of fixatives. DISCUSSION T h e present comparison of different fixation m e t h o d s demonstrates t h a t vascular perfusion with glutaraldehyde provides uniform fixation throughout the developing renal cortex. All other methods of applying the fixative caused variable qualities of fixation at different distances from the tissue surface. These observations are consistent with results obtained by the application of perfusion methods to other developing tissues (i, 22, 34). In the case of the kidney the uniform preservation of the developing cortex is significant since only under such conditions is it possible to m a k e a meaningful comparison between different developmental stages of the proximal tubules located at different levels below the kidney surface. Perfusion fixation should also be advantageous in histochemical studies since any diffusion of enzymes will be less by this m e t h o d t h a n in immersion fixation procedures where the fixative penetrates slowly (17). T h e present study demonstrates t h a t the
Fro. 7. Apical parts of a stage I tubule fixed with 6% glutaraldehyde in 0.01 M sodium cacodylate buffer. The cytoplasm is apparently swollen and many areas appear empty (arrows). Most mitochondria have a low and uneven electron density. Compare with Fig. 8 where the tissue is fixed in 6% glutaraldehyde but in 0.1 M sodium cacodylate buffer. The tissue was treated with uranyl acetate en bloc and embedded in Vestopal W. Sections stained with uranyl acetate and lead citrate. × 17 100. Fro. 8. Corresponding area as in Fig. 7 but the tissue is fixed with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer. The cytoplasm and mitochondria have a uniform electron density without evidence of swelling Embedding and staining as in Fig. 7. × 17 100.
FIXATION OF DEVELOPING PROXIMAL TUBULES
147
148
LARS LARSSON
FIG. 9. Basal parts of a proximal tubule in stage IV fixed with 6% glutaraldehyde in 0.01 M sodium cacodylate buffer. Note that the intercellular spaces are almost absent and that the cell membranes have formed long five-layered membrane complexes (arrows). Compare with Fig. 10 where the tissue is fixed with 6% glutaraldehyde but in 0.1 M sodium cacodylate buffer. The tissue was treated with uranyl acetate en bloc and embedded in Vestopal W. Sections stained with uranyl acetate and lead citrate. × 81 000. FIG. 10. Corresponding area as in Fig. 9 but the tissue is fixed with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer. Irregular intercellular spaces (IS) are seen and adjacent cell membranes are occasionally apposed forming five-layered membrane complexes (arrows). Embedding and staining as in Fig. 9. × 81 000. p e r f u s i o n f i x a t i o n m e t h o d w h i c h is s i m i l a r to t h a t e m p l o y e d for a d u l t r e n a l t u b u l e s (37) is also u s e f u l for d e v e l o p i n g r e n a l t u b u l e s p r o v i d e d t h a t t h e c o m p o s i t i o n of t h e f i x a t i v e is m o d i f i e d . T h e m o s t i m p o r tant modification concerns the concentrat i o n of g l u t a r a l d e h y d e w h i c h m u s t be r a i s e d to 6% in o r d e r to g e t s a t i s f a c t o r y
p r e s e r v a t i o n of t h e p l a s m a m e m b r a n e s in t h e first t w o d e v e l o p m e n t a l s t a g e s . In cont r a s t good f i x a t i o n of a d u l t p r o x i m a l tub u l e s is o b t a i n e d w i t h a c o n c e n t r a t i o n of g l u t a r a l d e h y d e of 1% or e v e n less (38). T h e r e q u i r e m e n t for a h i g h g l u t a r a l d e h y d e conc e n t r a t i o n m a y d e r i v e f r o m t h e p r e s e n c e in t h e d e v e l o p i n g r e n a l c o r t e x of a less r i c h
FIGS. 11-13. Different stages fixed by immersion with 4% formaldehyde and 5% glutaraldehyde in 0.1 M sodium cacodylate buffer and embedded in Vestopal. Sections stained with uranyl acetate and lead citrate. FIa. 11. Large intercellular spaces are seen in a renal vesicle or stage I (I) and a collecting tubule (CT) just below the renal capsule (RC). The intercellular spaces are less pronounced in stage III (III) deeper in the cortex. The cytoplasm is very electron dense in all cells. × 1700. FIa. 12. Part of stage I tubule. There are large intercellular spaces (IS) and the cytoplasm is very electron dense. It is difficult to identify the mitochondria (arrows). × 12 600. Fro. 13. Part of stage IV tubule. The intercellular spaces have approximately the same width as in tissue fixed with 6% glutaraldehyde in 0.1 M sodium cacodylate (compare with Fig. 10) but the mitochondria (M) are difficult to identify. × 27 900.
149
150
LARS LARSSON
vascular supply in the outer cortex t h a n in the inner cortex (35). In contrast in the adult kidney the vascular supply is rich t h r o u g h o u t cortex (14). Since glutaraldehyde has been shown to p e n e t r a t e faster at higher concentrations (18) the high concentration employed in this study m a y be efficient to overcome the disadvantage of the less extensive vasculature of the developing kidney. Recent studies have shown t h a t there m a y be an intercellular p a t h w a y of small molecules from one side of an epithelium to the other (11, 16). In any a t t e m p t to gain structural evidence for this p a t h w a y it is clearly i m p o r t a n t to ensure in the first instance t h a t the tissue is well preserved. This is e m p h a s i z e d by the present observation t h a t adjacent cell m e m b r a n e s were more frequently apposed in 0.01 M buffer t h a n in 0.1 M buffer. This shows t h a t the composition of the vehicle of the fixative influences the width of the intercellular spaces in developing as in adult renal tubules (38). It is therefore necessary to consider possible fixation alteration of the tissue before unequivocal conclusions can be drawn with regard to the a p p e a r a n c e of the intercellular spaces. T h e frequency and length of the basal five-layered m e m b r a n e complexes is dep e n d e n t upon the osmolality of the fixative since almost all these complexes disappeared when the tissue was fixed with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer. It is likely t h a t more m e m b r a n e complexes are being formed by the fixatives with a hypoosmolal buffer. It is unlikely t h a t they are true cell contacts since in other tissues these resist large changes in the osmolality of the vehicle of the fixative (5). Small variations in the composition of the vehicle of the fixative (0.07 M-0.1 M sodium cacodylate) have no obvious effects on the width of the intercellular spaces (28).
Tissue fixed with formaldehyde-containing fixatives showed, i n d e p e n d e n t of the
m e t h o d of application, wider intercellular spaces in early t h a n in late developmental stages. This m a y indicate t h a t early develo p m e n t a l stages are more sensitive to osmotic variations in the fixative t h a n the later stages in newborn rat kidney. Adult proximal tubule cells also seem to be more sensitive to the osmolality of the fixative t h a n proximal tubule cells of stages III and IV since vascular perfusion with 6% glutaraldehyde in 0.1 M sodium cacodylate showed very wide intercellular spaces in all proximal tubules t h r o u g h o u t the renal cortex in adult animals (30). These observations show t h a t a tissue at different develo p m e n t a l stages m a y react differently to a certain fixative and indicate the necessity to explore the effects of a fixative at each developmental stage under investigation. REFERENCES 1. ABRUNHOSA,R., J. Ultrastruct. Res. 41,176 (1972). 2. AOKI,A., Anat. Rec. 155, 339 (1966). 3. BENCOSME, S., STONE, R. S., LATTA, It., AND MADDEN, S. C., J. Biophys. Biochem. Cytol. 5,
508 (1959). 4. BOHMAN, S.-O., J. Ultrastruct. Res. 47, 329
(1974). 5. BRIGHTMAN,M. W., ANDREESE, W. S., J. Cell Biol.
40, 648 (1969). 6. BRODY, I., J. Ultrastruct. Res. 2, 482 (1959). 7. CALEY, D. W., AND MAXWELL, D. S., J. Comp. Neurol. 138, 31 (1970). 8. CLARK, S. L., g. Biophys. Biochem. Cytol. 3, 349
(1957). 9. COHEN,A. I., Develop. Biol. 3, 297 (1961). 10. DEHAAN,R. L., in DeHaan, R. L. and Ursprung, H. (Eds.), Organogenesis, p. 377. Holt, Rinehart and Winston, New York, 1965. 11. DmONA,D. R., Nature New Biol. 238, 179 (1972). 12. DUNCAN, D., Texas Rep. Biol. Med. 15, 367 (1957). 13. FAHIMI, H. D., AND DROCHMANS,P., J. Microsc. (Paris) 4, 725 (1965). 14. FOURMAN, J., AND MOFFAT, D. B., The blood
vessels of the kidney. Blackwell, Oxford, 1971. 15. FELDMAN,J. S., VAZQUEZ,J. J., ANDKURTZ, S. M., J. Biophys. Biochem. Cytol. ll, 365 (1961). 16. GIEBISCH,G., BOULPEAP,E. L., ANDWHITTEMBURY, G., Phil. Trans. Roy. Soc. Lond. B. 262, 175
(1971). 17. GOLDFISCHER,S., ESSNER, E., ANDNOVIKOFF,A. B., J. Histochern. Cytochem. 12, 72 (1964).
FIXATION OF DEVELOPING PROXIMAL TUBULES 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
32. 33. 34.
35. 36. 37. 38.
151
HOPWOOD,D., Histochem. J. 1, 323 (1969). (1966). HUBER, G. C., Am. J. Anat. 4, Suppl, 1 (1905). 39. MAUNSEACH,A. B., MADDEN,S. C., ANDLATTA,H., JOKELAINEN,P., Acta Anat. 52, Suppl, 47 (1963). J. Ultrastruct. Res. 6, 511 (1962). KALT, M. R., AND TANDLER, B., J. Ultrastruct. 40. NILSSON, S. E. G., J. Ultrastruct. Res. ll, 581 Res. 36, 633 (1971). (1964). KARLSSON,U., J. Ultrastruct. Res. 17, 158 (1967). 41. PEASE, D. C., Anat. Rec. 121, 701 (1955). KARNOVSKY,M. J., J. Cell Biol. 27, 137 A (1965). 42. PEASE, D. C., ibid. 142, 342 (1962). KAZIEMIERCZAK, J., Acta Pathol. Microbiol. 43. REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). Scand. Suppl. 218 (1971). 44. ROMEIS, B., Mikroskopische Technik, p. 31. O1KELLENBERGER,E., RYTER, A., ANDSI~CHAUD,J., J. denburg, Mtinchen, Wien, 1968. Biophys. Biochem. Cytol. 4, 671 (1958). 45. RYTER, A., ANDKELLENBERGER,E., J. Ultrastruct. KURTZ, S. M., Exp. Cell Res. 14, 355 (1958). Res. 2, 200 (1958). KURTZ, S. M., J. Ultrastruct. Res. 5, 468 (1961). 46. SABATINI,D. D., BENSCH,K., ANDBARRNETT,R. J., LARSSON,L., J. Ultrastruct. Res. 51, 119 (1975). J. Cell Biol. 17, 19 (i963). LARSSON,L., to be published. 47. SJI~STRAND,f. S., Electron Microscopy of Cells LARSSON,L., unpublished observations. and Tissues, Vol l, p. 144. Academic Press, New LARSSON, L., MAUNSEACH,A. B., SAXI~N, L., AND York, 1967. WARTIOVAARA,J., J. Ultrastruct. Res. 29, 570 48. SuzuKI, Y., J. Electron microsc. 6, 52 (1958). (1969). 49. WARTIOVAARA,J., Ann. Med. Exp. Biol. Fenn. 44, LARSSON,L., AND MAUI~SBACH,A. B., to be pub4O9 (1966). lished. 50. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, LEESON, T. S., Amer. J. Anat. 105, 165 (1959). 475 (1958). LESKES, A., SIEKEVITZ, P., AND PALADE, G. E., J. 51. WEBEER,W. A., ANDBLACKBOURN,J., Lab. Invest. Cell Biol. 49, 264 (1971). 23, 1 (1970). LJUNGQVIST,A., Acta Paediat. 52, 443 (1963). 52. VERNIER, R. L., AND BIRCH-ANDERSEN, A., J. LUFT, J., J. Biophys. Biochem. Cytol. 9, 409 Pediat. 60, 754 (1962). (1961). 53. YOSHIMURA,F., ANDNAKAMURA,M., Ohajimas Fol. MAUNSBACH,A. B., J. Ultrastruct. Res. 15, 242 Anat. Jap. 41, 121 (1965). (1966). 54. ZAMEONI,L., AND DE MARTINO, C., J. Ultrastruct. MAUNSBACH,A. B., J. Ultrastruct. Res. 15, 283 Res. 16, 399 (1966).