- Email: [email protected]
Differences between rat kidney proximal and distal epithelial cells as demonstrated with the cerium-based method for plasma membrane phosphatases
398
Abstracts oJ'The Netherlands SocieO, for Electron Microscopy & Royal Microscopical Socieo'
b r o k e n D e c e m e t m e m b r a n e s ; s e c o n d l y as an a r e a w i t h h i g h c e l l a c t i v i t y in o r d e r to r e p a i r the d a m a g e . In t h e a r e a w h e r e the i n t r a o c u l a r l e n s s t i l l w a s a t t a c h e d to t h e iris, the g r o w t h of f o r e i g n b o d y g i a n t c e l l s in c e l l u l a r m e m b r a n e s c o u l d b e o b s e r v e d o n the s u r f a c e of the a r t i f i c i a l lens. A t the i n g r o w t h a r e a of the i n e r t s t a i n l e s s s t e e l s u t u r e a s t r o n g t e n d e n c y to r e - e n d o t h e l i s a t i o n w a s o b s e r v a b l e , r e s u l t i n g in a c o m p l e t e r e g e n e r a t e d e n d o t h e l i u m a n d an i n t a c t Decemet membrane. E x a m i n i n g the d i f f e r e n t a r e a s e n a b l e d us to s t u d y the d y n a m ics of the r e p a i r i n g p r o c e s s . 1. M. J. Figueras, W. L. Jongebloed and J. G. Worst, Am. Intraocular Implant Soc. J. i0 (1984) 169-75. 2. H. A. Hanson, in: Scanning E.M. in Cell Biology and Medicine, Ed. K. Tanaka and T. Fujita (Excerpta Medica, Amsterdam, 1981), pp. 465-74. 3. J. Sugar, J. Burnett and S. L. Forstot, Am. J. Ophthalmol. 86 (1978) 157-61.
SIMULTANEOUS AES AND EDS MEASUREMENTS ON THIN SECTIONS OF SOILS S. H e n s t r a , E . B . A . B i s d o m * , O o i j * * , T.H. V i s s e r * *
W.J.
van
Technical and Physical Engineering R e s e a r c h S e r v i c e , P. O. B o x 356, NL-6700 AJ Wageningen; * S o i l S u r v e y I n s t i t u t e , P. O. B o x 98, NL-6700 AB Wageningen; **AKZO Research, Corporate Research, D e p a r t m e n t A r n h e m , The N e t h e r l a n d s
A E S m e a s u r e m e n t s on i n s u l a t i n g m a t e r i a l s a r e in m o s t c a s e s v e r y d i f f i c u l t o r t o t a l l y i m p o s s i b l e d u e to c h a r g i n g p h e n o m e n a on t h e s a m p l e s u r f a c e . For this reason previous AES analyses on thin s e c t i o n s of s o i l s w e r e v i r t u a l l y a l w a y s b o u n d to m i s h a p s . D u r i n g n e w e x p e r i m e n t s , the 20 u m thick specimen was mounted on a metal s u b s t r a t e a n d s p u t t e r e d w i t h a r g o n ions p r i o r to a n a l y s i s . These conditions, together with a very low probe current, could greatly reduce the charging eff e c t s t o an a c c e p t a b l e l e v e l d u r i n g A E S analysis. AES-spectra and -images could now easily be obtained from thin sections of soils. The use of a combinat i o n i n s t r u m e n t w i t h an e n e r g y - d i s p e r s i v e X-ray detector allowed simultaneous AES and EDS measurements f r o m the s a m e spot. D i f f e r e n c e s in s i z e a n d s h a p e o f the a n a l y z e d v o l u m e s in A E S a n d E D S a r e im-
p o r t a n t for t h e s t u d y o f the c h e m i c a l c o m p o s i t i o n of m a t e r i a l s in t h i n sect i o n s of s o i l s d u e to t h e i r e x t r e m e inhomogeneity even on a microscale. E.B.A. Bisdom, S. Henstra, H.W. Werner, P.R. Boudewijn, W.F. Knippenberg, H.A.M. De Grefte, J.M. Gourgout and H.N. Migeon, Quantitative analysis of trace and major elements in thin sections of soils with the secondary ion microscope (Cameca), Geoderma 30 (1983) 117.
DIFFERENCES BETWEEN RAT KIDNEY PROXIMAL A N D D I S T A L E P I T H E L I A L C E L L S AS D E M O N STRATED WITH THE CERIUM-BASED METHOD FOR PLASMA MEMBRANE PHOSPHATASES C. E. M. J.
Hulstaert, Hardonk*
D. K a l i c h a r a n
and
C e n t r e for M E d i c a l E l e c t r o n M i c r o s copy, University of Gronin~en, Oostersingel 69/2; * D e p a r t m e n t o f Pathology, University of Groningen, Oostersingel 63, 9 7 1 3 EZ G r o n i n ~ e n , The N e t h e r l a n d s
T h e c e r i u m - b a s e d m e t h o d for the d e m o n s t r a t i o n of p h o s p h a t a s e s l e a d s to a m o r e p r e c i s e l o c a l i z a t i o n o~ the r e a c t i o n n r o d u c t t h a n the l e a d - b a s e d m e t h o d of H u l s t a e r t et al. I a n d R o b i n s o n a n d Karnovsk}r. 2 T h e c e r i u m - b a s e d m e t h o d is a l s o c a p a b l e of d e m o n s t r a t i n ~ d i f f e r e n ces in p l a s m a m e m b r a n e p h o s p h a t a s e activities between proximal and distal e p i t h e l i a l c e l l s in the r a t k i d n e y . K i d n e v s of m a l e W i s t a r r a t s w e r e f i r s t p e r f u s e d v i a the a r t e r i a r e n a l i s 3 for 1 m i n w i t h 2% p o l y v i n y l p y r r o l iOone, 75 m M N a N O 2 in O . i M c a c o d y l a t e b u f f e r pH 7.4, s u b s e q u e n t l y for 3 m i n w i t h 2% m o n o m e r i c g l u t a r a l d e h y d e in 0 . i M c a c o d y l a t e b u f f e r , pH 7.4, a n d f i n a l l y w i t h 6.8% s a c c h a r o s e in 0 . i M c a c o d y l a t e b u f f e r , pH 7.4, to r e m o v e the f i x a t i v e . A f t e r a p r e i n c u b a t i o n for 1 h o u r in m e d i u m w i t h o u t s u b s t r a t e , 30 m v i b r a t o m e s e c t i o n s w e r e i n c u b a t e d (30 min) for a l k a l i n e p h o s p h a t a s e a n d nucleoside di- and triphosphatases. The m e d i u m for the d e m o n s t r a t i o n o f a l k a l i n e p h o s p h a t a s e c o n s i s t e d of 1 m M C e C I 3 , 4 mM MgCI2, 7 mM Na-B-glycerophosphate a n d 70 m M T r i s - m a l e a t e b u f f e r , pH 9.0. T h e m e d i a for t h e d e m o n s t r a t i o n of nucleoside di- and triphosphatases cons i s t e d of 1 m M C e C I 3, 5 m M M g ( N O 3 ) 2, 2.3 m M (ATP, ADP, UTP, U D P or ITP), a n d 70 m M T r i s - m a l e a t e b u f f e r , p H 7.2. A f t e r r i n s i n g o v e r n i g h t at 4°C in 6.8% s a c c h a r o s e in O . i M c a c o d y l a t e b u f f e r , pH 6.0,
Abstracts of The Netherlands Society for Electron Microscopy & Royal Microscopical Society
tO r e m o v e the i n c u b a t i o n m e d i u m and nonspeci f i c p r e c i p i t a t e s , the v i b r a t o m e s ecti o n s w e r e p o s t f i x e d for 2 hours at 4oc in 1% OsO4, 1.5% K4Fe(CN) 6 in O.i M c a c o d y l a t e buffer, pH 7.4. A f t e r dehyd r a t i o n in an a l c o h o l series the sections were e m b e d d e d in Epon. The p r o x i m a l e p i t h e l i a l cells exhibited clear d i f f e r e n c e s in a c t i v i t y at their m i c r o v i l l i for all p h o s p h a t a s e s investigated. F u r t h e r m o r e , the d i s t a l e p i t h e l i a l cells showed d i f f e r e n c e s in a c t i v i t y at their m i c r o v i l l i w h e n incub ated for A T P a s e and UTPase. These d i f f e r e n c e s could be a r e f l e c t i o n of a f u n c t i o n a l or a phase d i f f e r e n c e b e t w e e n these cells. 1. 2. 3.
C. E. Hulstaert, D. Kalicharan and M. J. Hardonk, Histochem. 78 (1983) 71. J. M. Robinson and M. J. Karnovsky, Histochem. Cytochem. 31 (1983) 1197. A. B. Maunsbach, J. Ultrastruct. Res. 15 (1966) 242.
399
nitrogen. S u b s e q u e n t l y , the tissue blocks (0.5 x 0.5 x 0.5 cm) were t h a w e d in 6.8% s a c c h a r o s e in O.i M c a c o d y l a t e buffer, pH 7.4, and 30 ~m thick sections w e r e cut w i t h a vibratome. The v i b r a t o m e sections were prei n c u b a t e d for 1 hour at 4oc in the i n c u b a t i o n m e d i u m (I m M CeCI3, 2 m M disodium-glucose-6-phosphate, 80 mM T r i s - m a l e a t e buffer, pH 6.5) f o l l o w e d by i n c u b a t i o n for 30 min at 37oc in the same medium. F r e e z i n g liver tissue in m e l t i n g n i t r o g e n after fixation a p p a r e n t l y makes the p l a s m a m e m b r a n e c o m p l e t e l y p e r m e a b l e for the p e n e t r a t i o n of the m e d i u m constituents and also removes all h i n d r a n ces for the p e n e t r a t i o n of these constituents into the cytoplasm. 1.
c. E. Hulstaert, D. Kalicharan and M. J. Hardonk, Histochem. 78 (1983) 71.
IN VIVO AND IN V I T R O R E M I N E R A L I Z A T I O N OF BOVINE E N A M E L COMPARED, A SEM STUDY P R E V E N T I O N OF THE P E N E T R A T I O N H I N D R A N C E IN C E R I U M - B A S E D G L U C O S E - 6 - P H O S P H A T A S E C Y T O C H E M I S T R Y BY F R E E Z I N G T I S S U E IN MELTING NITROGEN C. E. Hulstaert, M. J. H a r d o n k *
D. K a l i c h a r a n
and
centre for Medical Electron Microscopy, U n i v e r s i t y of Groningen, Oostersingel 69/2; *Department of Pathology, University of Groningen, oostersingel 63, 9 7 1 3 EZ G r o n i n g e n , The N e t h e r l a n d s
The c e r i u m - b a s e d m e t h o d gives excellent r e s u l t s for the d e m o n s t r a t i o n of p l a s m a m e m b r a n e p h o s p h a t a s e activities. 1 However, it a p p e a r e d that the localization of the i n t r a c e l l u l a r l y l o c a l i z e d g l u c o s e - 6 - p h o s p h a t a s e is h a m p e r e d by p e n e t r a t i o n p r o b l e m s of m e d i u m constituents, a l t h o u g h 30 ~m liver v i b r a t o m e secti o n s were p r e i n c u b a t e d for 1 hour in m e d i u m w i t h o u t substrate. Examination of p e r p e n d i c u l a r s e c t i o n e d vibratome s e c t i o n s r e v e a l e d that c o n s i s t e n t l y more r e a c t i o n p r o d u c t was p r e s e n t near the surface w h i l e in the centre the r e a c t i o n p r o d u c t was less, or even absent. B e s i d e s the p l a s m a m e m b r a n e , also the c y t o p l a s m appears to h a m p e r the p e n e t r a t i o n of the m e d i u m constituents. A f t e r several e x p e r i m e n t a l trials w e found that the p e n e t r a t i o n h i n d r a n c e could be p r e v e n t e d w h e n g l u t a r a l d e h y d e fixed liver tissue was frozen in m e l t i n g
W. L. J o n g e b l o e d , J. Arends*
T.B.F.M.
Gelhard*
and
centre for Medical Electron Microscopy, S t a t e U n i v e r s i t y , Oostersingel 69/2, 9 7 1 3 EZ G r o n i n g e n , The Netherlands; *Dental School, State University, Ant. D e u s i n g l a a n i, 9 7 1 3 AV Groningen, The N e t h e r l a n d s
Introduction: R e m i n e r a l i z a t i o n of enamel defects, e.g., r e d e p o s i t i o n of apatite material, p a r t i c u l a r l y in vivo, is of c o n s i d e r a b l e i m p o r t a n c e in dentistry. M a t e r i a l s and methods: In vivo: polished, softened, sterilized, bovine enamel slabs were p l a c e d in the prosthesis of p a t i e n t s for periods up to 3 months, under various F - c o n d i t i o n s up to 15OO ppm, for details see G e l h a r d et al. I In vitro: p o l i s h e d and via a w i n d o w p a r t l y d e c a l c i f i e d b o v i n e elements were r e m i n e r a l i z e d in a s a t u r a t e d h y d r o x y a p a t i t e s o l u t i o n for 14 days. For SEM o b s e r v a t i o n s the samples were, after washing, fixed (2% GA + 1% OsO4 s o l u t i o n s in O . i M c a c o d y l a t e buffer), washed, d e h y d r a t e d in ethanol, c r i t i c a l p o i n t - d r i e d with CO2, c l e a v e d and finally s p u t t e r - c o a t e d (iO nm Au-Pd). S p e c i m e n s w e r e e x a m i n e d in a JEOL SEM, type 35C, o p e r a t e d at 25-35 kV. Results and c o n c l u s i o n s : (i) Remine r a l i z a t i o n is m a i n l y o b s e r v a b l e in i n t e r p r i s m a t i c areas. (2) The in vivo
Abstracts oJ'The Netherlands SocieO, for Electron Microscopy & Royal Microscopical Socieo'
b r o k e n D e c e m e t m e m b r a n e s ; s e c o n d l y as an a r e a w i t h h i g h c e l l a c t i v i t y in o r d e r to r e p a i r the d a m a g e . In t h e a r e a w h e r e the i n t r a o c u l a r l e n s s t i l l w a s a t t a c h e d to t h e iris, the g r o w t h of f o r e i g n b o d y g i a n t c e l l s in c e l l u l a r m e m b r a n e s c o u l d b e o b s e r v e d o n the s u r f a c e of the a r t i f i c i a l lens. A t the i n g r o w t h a r e a of the i n e r t s t a i n l e s s s t e e l s u t u r e a s t r o n g t e n d e n c y to r e - e n d o t h e l i s a t i o n w a s o b s e r v a b l e , r e s u l t i n g in a c o m p l e t e r e g e n e r a t e d e n d o t h e l i u m a n d an i n t a c t Decemet membrane. E x a m i n i n g the d i f f e r e n t a r e a s e n a b l e d us to s t u d y the d y n a m ics of the r e p a i r i n g p r o c e s s . 1. M. J. Figueras, W. L. Jongebloed and J. G. Worst, Am. Intraocular Implant Soc. J. i0 (1984) 169-75. 2. H. A. Hanson, in: Scanning E.M. in Cell Biology and Medicine, Ed. K. Tanaka and T. Fujita (Excerpta Medica, Amsterdam, 1981), pp. 465-74. 3. J. Sugar, J. Burnett and S. L. Forstot, Am. J. Ophthalmol. 86 (1978) 157-61.
SIMULTANEOUS AES AND EDS MEASUREMENTS ON THIN SECTIONS OF SOILS S. H e n s t r a , E . B . A . B i s d o m * , O o i j * * , T.H. V i s s e r * *
W.J.
van
Technical and Physical Engineering R e s e a r c h S e r v i c e , P. O. B o x 356, NL-6700 AJ Wageningen; * S o i l S u r v e y I n s t i t u t e , P. O. B o x 98, NL-6700 AB Wageningen; **AKZO Research, Corporate Research, D e p a r t m e n t A r n h e m , The N e t h e r l a n d s
A E S m e a s u r e m e n t s on i n s u l a t i n g m a t e r i a l s a r e in m o s t c a s e s v e r y d i f f i c u l t o r t o t a l l y i m p o s s i b l e d u e to c h a r g i n g p h e n o m e n a on t h e s a m p l e s u r f a c e . For this reason previous AES analyses on thin s e c t i o n s of s o i l s w e r e v i r t u a l l y a l w a y s b o u n d to m i s h a p s . D u r i n g n e w e x p e r i m e n t s , the 20 u m thick specimen was mounted on a metal s u b s t r a t e a n d s p u t t e r e d w i t h a r g o n ions p r i o r to a n a l y s i s . These conditions, together with a very low probe current, could greatly reduce the charging eff e c t s t o an a c c e p t a b l e l e v e l d u r i n g A E S analysis. AES-spectra and -images could now easily be obtained from thin sections of soils. The use of a combinat i o n i n s t r u m e n t w i t h an e n e r g y - d i s p e r s i v e X-ray detector allowed simultaneous AES and EDS measurements f r o m the s a m e spot. D i f f e r e n c e s in s i z e a n d s h a p e o f the a n a l y z e d v o l u m e s in A E S a n d E D S a r e im-
p o r t a n t for t h e s t u d y o f the c h e m i c a l c o m p o s i t i o n of m a t e r i a l s in t h i n sect i o n s of s o i l s d u e to t h e i r e x t r e m e inhomogeneity even on a microscale. E.B.A. Bisdom, S. Henstra, H.W. Werner, P.R. Boudewijn, W.F. Knippenberg, H.A.M. De Grefte, J.M. Gourgout and H.N. Migeon, Quantitative analysis of trace and major elements in thin sections of soils with the secondary ion microscope (Cameca), Geoderma 30 (1983) 117.
DIFFERENCES BETWEEN RAT KIDNEY PROXIMAL A N D D I S T A L E P I T H E L I A L C E L L S AS D E M O N STRATED WITH THE CERIUM-BASED METHOD FOR PLASMA MEMBRANE PHOSPHATASES C. E. M. J.
Hulstaert, Hardonk*
D. K a l i c h a r a n
and
C e n t r e for M E d i c a l E l e c t r o n M i c r o s copy, University of Gronin~en, Oostersingel 69/2; * D e p a r t m e n t o f Pathology, University of Groningen, Oostersingel 63, 9 7 1 3 EZ G r o n i n ~ e n , The N e t h e r l a n d s
T h e c e r i u m - b a s e d m e t h o d for the d e m o n s t r a t i o n of p h o s p h a t a s e s l e a d s to a m o r e p r e c i s e l o c a l i z a t i o n o~ the r e a c t i o n n r o d u c t t h a n the l e a d - b a s e d m e t h o d of H u l s t a e r t et al. I a n d R o b i n s o n a n d Karnovsk}r. 2 T h e c e r i u m - b a s e d m e t h o d is a l s o c a p a b l e of d e m o n s t r a t i n ~ d i f f e r e n ces in p l a s m a m e m b r a n e p h o s p h a t a s e activities between proximal and distal e p i t h e l i a l c e l l s in the r a t k i d n e y . K i d n e v s of m a l e W i s t a r r a t s w e r e f i r s t p e r f u s e d v i a the a r t e r i a r e n a l i s 3 for 1 m i n w i t h 2% p o l y v i n y l p y r r o l iOone, 75 m M N a N O 2 in O . i M c a c o d y l a t e b u f f e r pH 7.4, s u b s e q u e n t l y for 3 m i n w i t h 2% m o n o m e r i c g l u t a r a l d e h y d e in 0 . i M c a c o d y l a t e b u f f e r , pH 7.4, a n d f i n a l l y w i t h 6.8% s a c c h a r o s e in 0 . i M c a c o d y l a t e b u f f e r , pH 7.4, to r e m o v e the f i x a t i v e . A f t e r a p r e i n c u b a t i o n for 1 h o u r in m e d i u m w i t h o u t s u b s t r a t e , 30 m v i b r a t o m e s e c t i o n s w e r e i n c u b a t e d (30 min) for a l k a l i n e p h o s p h a t a s e a n d nucleoside di- and triphosphatases. The m e d i u m for the d e m o n s t r a t i o n o f a l k a l i n e p h o s p h a t a s e c o n s i s t e d of 1 m M C e C I 3 , 4 mM MgCI2, 7 mM Na-B-glycerophosphate a n d 70 m M T r i s - m a l e a t e b u f f e r , pH 9.0. T h e m e d i a for t h e d e m o n s t r a t i o n of nucleoside di- and triphosphatases cons i s t e d of 1 m M C e C I 3, 5 m M M g ( N O 3 ) 2, 2.3 m M (ATP, ADP, UTP, U D P or ITP), a n d 70 m M T r i s - m a l e a t e b u f f e r , p H 7.2. A f t e r r i n s i n g o v e r n i g h t at 4°C in 6.8% s a c c h a r o s e in O . i M c a c o d y l a t e b u f f e r , pH 6.0,
Abstracts of The Netherlands Society for Electron Microscopy & Royal Microscopical Society
tO r e m o v e the i n c u b a t i o n m e d i u m and nonspeci f i c p r e c i p i t a t e s , the v i b r a t o m e s ecti o n s w e r e p o s t f i x e d for 2 hours at 4oc in 1% OsO4, 1.5% K4Fe(CN) 6 in O.i M c a c o d y l a t e buffer, pH 7.4. A f t e r dehyd r a t i o n in an a l c o h o l series the sections were e m b e d d e d in Epon. The p r o x i m a l e p i t h e l i a l cells exhibited clear d i f f e r e n c e s in a c t i v i t y at their m i c r o v i l l i for all p h o s p h a t a s e s investigated. F u r t h e r m o r e , the d i s t a l e p i t h e l i a l cells showed d i f f e r e n c e s in a c t i v i t y at their m i c r o v i l l i w h e n incub ated for A T P a s e and UTPase. These d i f f e r e n c e s could be a r e f l e c t i o n of a f u n c t i o n a l or a phase d i f f e r e n c e b e t w e e n these cells. 1. 2. 3.
C. E. Hulstaert, D. Kalicharan and M. J. Hardonk, Histochem. 78 (1983) 71. J. M. Robinson and M. J. Karnovsky, Histochem. Cytochem. 31 (1983) 1197. A. B. Maunsbach, J. Ultrastruct. Res. 15 (1966) 242.
399
nitrogen. S u b s e q u e n t l y , the tissue blocks (0.5 x 0.5 x 0.5 cm) were t h a w e d in 6.8% s a c c h a r o s e in O.i M c a c o d y l a t e buffer, pH 7.4, and 30 ~m thick sections w e r e cut w i t h a vibratome. The v i b r a t o m e sections were prei n c u b a t e d for 1 hour at 4oc in the i n c u b a t i o n m e d i u m (I m M CeCI3, 2 m M disodium-glucose-6-phosphate, 80 mM T r i s - m a l e a t e buffer, pH 6.5) f o l l o w e d by i n c u b a t i o n for 30 min at 37oc in the same medium. F r e e z i n g liver tissue in m e l t i n g n i t r o g e n after fixation a p p a r e n t l y makes the p l a s m a m e m b r a n e c o m p l e t e l y p e r m e a b l e for the p e n e t r a t i o n of the m e d i u m constituents and also removes all h i n d r a n ces for the p e n e t r a t i o n of these constituents into the cytoplasm. 1.
c. E. Hulstaert, D. Kalicharan and M. J. Hardonk, Histochem. 78 (1983) 71.
IN VIVO AND IN V I T R O R E M I N E R A L I Z A T I O N OF BOVINE E N A M E L COMPARED, A SEM STUDY P R E V E N T I O N OF THE P E N E T R A T I O N H I N D R A N C E IN C E R I U M - B A S E D G L U C O S E - 6 - P H O S P H A T A S E C Y T O C H E M I S T R Y BY F R E E Z I N G T I S S U E IN MELTING NITROGEN C. E. Hulstaert, M. J. H a r d o n k *
D. K a l i c h a r a n
and
centre for Medical Electron Microscopy, U n i v e r s i t y of Groningen, Oostersingel 69/2; *Department of Pathology, University of Groningen, oostersingel 63, 9 7 1 3 EZ G r o n i n g e n , The N e t h e r l a n d s
The c e r i u m - b a s e d m e t h o d gives excellent r e s u l t s for the d e m o n s t r a t i o n of p l a s m a m e m b r a n e p h o s p h a t a s e activities. 1 However, it a p p e a r e d that the localization of the i n t r a c e l l u l a r l y l o c a l i z e d g l u c o s e - 6 - p h o s p h a t a s e is h a m p e r e d by p e n e t r a t i o n p r o b l e m s of m e d i u m constituents, a l t h o u g h 30 ~m liver v i b r a t o m e secti o n s were p r e i n c u b a t e d for 1 hour in m e d i u m w i t h o u t substrate. Examination of p e r p e n d i c u l a r s e c t i o n e d vibratome s e c t i o n s r e v e a l e d that c o n s i s t e n t l y more r e a c t i o n p r o d u c t was p r e s e n t near the surface w h i l e in the centre the r e a c t i o n p r o d u c t was less, or even absent. B e s i d e s the p l a s m a m e m b r a n e , also the c y t o p l a s m appears to h a m p e r the p e n e t r a t i o n of the m e d i u m constituents. A f t e r several e x p e r i m e n t a l trials w e found that the p e n e t r a t i o n h i n d r a n c e could be p r e v e n t e d w h e n g l u t a r a l d e h y d e fixed liver tissue was frozen in m e l t i n g
W. L. J o n g e b l o e d , J. Arends*
T.B.F.M.
Gelhard*
and
centre for Medical Electron Microscopy, S t a t e U n i v e r s i t y , Oostersingel 69/2, 9 7 1 3 EZ G r o n i n g e n , The Netherlands; *Dental School, State University, Ant. D e u s i n g l a a n i, 9 7 1 3 AV Groningen, The N e t h e r l a n d s
Introduction: R e m i n e r a l i z a t i o n of enamel defects, e.g., r e d e p o s i t i o n of apatite material, p a r t i c u l a r l y in vivo, is of c o n s i d e r a b l e i m p o r t a n c e in dentistry. M a t e r i a l s and methods: In vivo: polished, softened, sterilized, bovine enamel slabs were p l a c e d in the prosthesis of p a t i e n t s for periods up to 3 months, under various F - c o n d i t i o n s up to 15OO ppm, for details see G e l h a r d et al. I In vitro: p o l i s h e d and via a w i n d o w p a r t l y d e c a l c i f i e d b o v i n e elements were r e m i n e r a l i z e d in a s a t u r a t e d h y d r o x y a p a t i t e s o l u t i o n for 14 days. For SEM o b s e r v a t i o n s the samples were, after washing, fixed (2% GA + 1% OsO4 s o l u t i o n s in O . i M c a c o d y l a t e buffer), washed, d e h y d r a t e d in ethanol, c r i t i c a l p o i n t - d r i e d with CO2, c l e a v e d and finally s p u t t e r - c o a t e d (iO nm Au-Pd). S p e c i m e n s w e r e e x a m i n e d in a JEOL SEM, type 35C, o p e r a t e d at 25-35 kV. Results and c o n c l u s i o n s : (i) Remine r a l i z a t i o n is m a i n l y o b s e r v a b l e in i n t e r p r i s m a t i c areas. (2) The in vivo