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Purification of glutaraldehyde by vacuum fractional distillation
Abstracts of The Netherlands Society of Electron Microscopy Annual Meeting s i s t i n g of f l a t t e n e d s u p e r f i c i a l c e l l s , are w e a k l y s t a i n e d . T a n n i c acid c o u l d n o t b e d e m o n s t r a t e d o n t h e s u r f a c e o f the c a r c i n o m a in s i t u c e l l s and the i n v a d i n g squamous carcinoma cells. It s e e m s t h a t s u r f a c e s t a i n i n g w i t h t a n n i c a c i d is a p r o m i s i n g a p p r o a c h i n d i s c r i m i n a t i n g n o r m a l , p r e - m a l i g n a n t and malignant ectocervical epithelia. 1.
P. Kenemans et al. in Biology of the Cancer Cell, ed. K. Letnansky (Kugler Publ., Amsterdam, 1980). 2. J. H. M. Davina et al., Scanning Electron Microscopy, vol. 3, pp. 37-48 (1981). 3. V. Mizuhira et al., Acta Histochem. Cytochem. 5 (1972) 233-36. Supported by the Dutch Cancer Society (number NUKC-SMM 79~7)
in an o i l b a t h . The f i r s t d i s t i l l a t e , c o l l e c t e d a t a t e m p e r a t u r e o f 2 0 ° C to 40°C in a b o u t two h o u r s a n d c o n t a i n i n g i m p u r i t i e s , was d i s c a r d e d . S u b s e q u e n t l y , the p u r i f i e d g l u t a r a l d e h y d e w i t h a Pi < 0 . 2 0 w a s c o l l e c t e d at a t e m p e r a t u r e o f 8 0 o c in a b o u t o n e - a n d - a - h a l f h o u r s . i. 2. 3.
P U R I F I C A T I O N OF G L U T A R A L D E H Y D E B Y VACUUM FRACTIONAL DISTILLATION D i j k , J. A. O o s t e r b a a n , E. H u l s t a e r t 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 c o p y , U n i v e r s i t y of G r o n i n g e n , G r o n i n g e n , The N e t h e r l a n d s
The e m p l o y m e n t of m o n o m e r i c g l u t a r a l d e h y d e as a f i x a t i v e is a d v a n t a g e o u s f o r e n z y m e c y t o c h e m i s t r y as t h e e n z y m e a c t i v i t y is b e t t e r r e t a i n e d t h a n w i t h p o l y m e r ic g l u t a r a l d e h y d e . I F a h i m i and D r o c h m a n s 2 d e v e l o p e d a m e t h o d to o b t a i n m o n o m e r i c g l u t a r a l d e h y d e by d i s t i l l a t i o n . A c c o r d i n g to t h e i r m e t h o d the g l u t a r a l d e h y d e s o l u t i o n h a d to b e d i s t i l l e d t w i c e in o r d e r to o b t a i n a h i g h c o n c e n t r a t i o n of m o n o m e r s . We h a v e d e v e l o p e d a m e t h o d w h i c h y i e l d s a h i g h c o n c e n t r a t i o n of m o n o m e r s a f t e r o n l y one d i s t i l l a t i o n . T h e p u r i f i c a t i o n o f g l u t a r a l d e h y d e is e x p r e s s e d as t h e p u r i f i c a t i o n i n d e x , Pi
=
e x t i n c t i o n at e x t i n c t i o n at
235 280
nm nm"
A t 235 n m t h e i m p u r i t i e s a n d p o l y m e r s a r e m e a s u r e d w h i l e at 280 n m t h e m o n o m e r s a r e measured. 3 F o r the d i s t i l l a t i o n p r o c e s s we h a v e u s e d an a d i a b a t i c f r a c t i o n a t i o n c o l u m n to r e d u c e t h e h e a t e x c h a n g e . T h e i n t e r i o r o f t h e f r a c t i o n a t i o n c o l u m n is f i l l e d w i t h s m a l l g l a s s c y l i n d e r s , l e a d i n g to a l a r g e s u r f a c e i n c r e a s e , thus f a c i l i t a t ing the p u r i f i c a t i o n of the g l u t a r a l d e hyde. T h e v a p o u r s c o n d e n s e in a v e r t i c a l c o o l i n g u n i t a n d t h e d i s t i l l a t e is l e d to t w o f l a s k s . T h e d i s t i l l a t i o n is c a r r i e d out u n d e r v a c u u m at a p r e s s u r e o f 10 m m o f m e r c u r y . A n a m o u n t o f 650 m l 25% g l u t a r a l d e h y d e s o l u t i o n w i t h a Pi > 10 w a s d i s t i l l e d each time, u s i n g a flask h e a t e d
P. J. Anderson, J. Histochem. Cytochem. 15 (1967) 652. H. D. Fahimi, P. Drochmans, J. Microscopie 4 (1965) 725. E. A. Robertson, R. L. Schultz, J. Ultrastr. Res. 30 (1969)
QUANTITATIVE A C Q U I S I T I O N AND E V A L U A T I O N OF E L E C T R O N M I C R O G R A P H Y U S I N G A S T E M A.
F. C.
421
Engel E i o z e n t r u m der U n i v e r s i t a t E a s e l , Abteilung Mikrobiologie, Klingelbergs t r a s s e 70, C H - 4 0 5 6 E a s e l , S w i t z e r l a n d
P r e p a r a t i o n and o b s e r v a t i o n of b i o l o g i c a l samples with e l e c t r o n microscopical m e t h o d s i n e v i t a b l y l e a d to d i s t o r t i o n s and o t h e r k i n d s of n o i s e , w h i c h o b s c u r e the r e p r e s e n t a t i o n of the true s t r u c t u r e . D i g i t a l a c q u i s i t i o n of e l e c t r o n micrographs u s i n g a STEM provides images in a f o r m a t r e a d y f o r d i g i t a l p r o c e s s i n g , w h i c h in t u r n p e r m i t s e x t r a c t i o n o f b i o logically useful structural information from n o i s y images.
INTRAMEMBRANEOUS PARTICLES ASSOCIATED WITH S U R F A C E IMMUNOGLOBULIN RECEPTORS IN T H E P L A S M A M E M B R A N E O F B L Y M P H O C Y T E S C.
De G r o o t ,
W.
Leene
L a b o r a t o r y of H i s t o l o g y and Cell Biol o g y , U n i v e r s i t y of A m s t e r d a m , 1 e C o n e t . H u y g e n s s t r a a t 20, 1054 BW A m s t e r d a m , The N e t h e r l a n d s
C o n v i n c i n g e v i d e n c e is a c c u m u l a t i n g t h a t s u r f a c e i m m u n o g l o b u l i n (sIg) r e c e p tor m o l e c u l e s of B l y m p h o c y t e s p o s s e s s two " e x t r a " h y d r o p h o b i c a m i n o a c i d seq u e n c e s w h e n c o m p a r e d w i t h s e c r e t e d imm u n o g l o b u l i n s . I-3 These hydrophobic p e p t i d e s a r e b e l i e v e d to f o r m a t r a n s m e m b r a n e s t r u c t u r e t h a t p l a y s an i m p o r t a n t r o l e in t h e t r a n s f e r o f a s i g n a l to the c y t o p l a s m . In t h e p r e s e n t s t u d y w e h a v e t r i e d b y f r e e z e - f r a c t u r i n g to v i s u a l i z e i n t r a m e m b r a n e o u s p a r t i c l e s (IMPs) s p e c i f i c a l l y a s s o c i a t e d w i t h sIg r e c e p t o r m o l e c u l e s . s I g r e c e p t o r s .on m o u s e a n d r a b b i t s p l e e n B l y m p h o c y t e s w e r e r e d i s t r i b u t e d by a n t i b o d i e s i n t o p a t c h e s and g l u t a r a l d e h y d e f i x e d c e l l s f r o z e n in 35% g l y c e r o l w e r e
P. Kenemans et al. in Biology of the Cancer Cell, ed. K. Letnansky (Kugler Publ., Amsterdam, 1980). 2. J. H. M. Davina et al., Scanning Electron Microscopy, vol. 3, pp. 37-48 (1981). 3. V. Mizuhira et al., Acta Histochem. Cytochem. 5 (1972) 233-36. Supported by the Dutch Cancer Society (number NUKC-SMM 79~7)
in an o i l b a t h . The f i r s t d i s t i l l a t e , c o l l e c t e d a t a t e m p e r a t u r e o f 2 0 ° C to 40°C in a b o u t two h o u r s a n d c o n t a i n i n g i m p u r i t i e s , was d i s c a r d e d . S u b s e q u e n t l y , the p u r i f i e d g l u t a r a l d e h y d e w i t h a Pi < 0 . 2 0 w a s c o l l e c t e d at a t e m p e r a t u r e o f 8 0 o c in a b o u t o n e - a n d - a - h a l f h o u r s . i. 2. 3.
P U R I F I C A T I O N OF G L U T A R A L D E H Y D E B Y VACUUM FRACTIONAL DISTILLATION D i j k , J. A. O o s t e r b a a n , E. H u l s t a e r t 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 c o p y , U n i v e r s i t y of G r o n i n g e n , G r o n i n g e n , The N e t h e r l a n d s
The e m p l o y m e n t of m o n o m e r i c g l u t a r a l d e h y d e as a f i x a t i v e is a d v a n t a g e o u s f o r e n z y m e c y t o c h e m i s t r y as t h e e n z y m e a c t i v i t y is b e t t e r r e t a i n e d t h a n w i t h p o l y m e r ic g l u t a r a l d e h y d e . I F a h i m i and D r o c h m a n s 2 d e v e l o p e d a m e t h o d to o b t a i n m o n o m e r i c g l u t a r a l d e h y d e by d i s t i l l a t i o n . A c c o r d i n g to t h e i r m e t h o d the g l u t a r a l d e h y d e s o l u t i o n h a d to b e d i s t i l l e d t w i c e in o r d e r to o b t a i n a h i g h c o n c e n t r a t i o n of m o n o m e r s . We h a v e d e v e l o p e d a m e t h o d w h i c h y i e l d s a h i g h c o n c e n t r a t i o n of m o n o m e r s a f t e r o n l y one d i s t i l l a t i o n . T h e p u r i f i c a t i o n o f g l u t a r a l d e h y d e is e x p r e s s e d as t h e p u r i f i c a t i o n i n d e x , Pi
=
e x t i n c t i o n at e x t i n c t i o n at
235 280
nm nm"
A t 235 n m t h e i m p u r i t i e s a n d p o l y m e r s a r e m e a s u r e d w h i l e at 280 n m t h e m o n o m e r s a r e measured. 3 F o r the d i s t i l l a t i o n p r o c e s s we h a v e u s e d an a d i a b a t i c f r a c t i o n a t i o n c o l u m n to r e d u c e t h e h e a t e x c h a n g e . T h e i n t e r i o r o f t h e f r a c t i o n a t i o n c o l u m n is f i l l e d w i t h s m a l l g l a s s c y l i n d e r s , l e a d i n g to a l a r g e s u r f a c e i n c r e a s e , thus f a c i l i t a t ing the p u r i f i c a t i o n of the g l u t a r a l d e hyde. T h e v a p o u r s c o n d e n s e in a v e r t i c a l c o o l i n g u n i t a n d t h e d i s t i l l a t e is l e d to t w o f l a s k s . T h e d i s t i l l a t i o n is c a r r i e d out u n d e r v a c u u m at a p r e s s u r e o f 10 m m o f m e r c u r y . A n a m o u n t o f 650 m l 25% g l u t a r a l d e h y d e s o l u t i o n w i t h a Pi > 10 w a s d i s t i l l e d each time, u s i n g a flask h e a t e d
P. J. Anderson, J. Histochem. Cytochem. 15 (1967) 652. H. D. Fahimi, P. Drochmans, J. Microscopie 4 (1965) 725. E. A. Robertson, R. L. Schultz, J. Ultrastr. Res. 30 (1969)
QUANTITATIVE A C Q U I S I T I O N AND E V A L U A T I O N OF E L E C T R O N M I C R O G R A P H Y U S I N G A S T E M A.
F. C.
421
Engel E i o z e n t r u m der U n i v e r s i t a t E a s e l , Abteilung Mikrobiologie, Klingelbergs t r a s s e 70, C H - 4 0 5 6 E a s e l , S w i t z e r l a n d
P r e p a r a t i o n and o b s e r v a t i o n of b i o l o g i c a l samples with e l e c t r o n microscopical m e t h o d s i n e v i t a b l y l e a d to d i s t o r t i o n s and o t h e r k i n d s of n o i s e , w h i c h o b s c u r e the r e p r e s e n t a t i o n of the true s t r u c t u r e . D i g i t a l a c q u i s i t i o n of e l e c t r o n micrographs u s i n g a STEM provides images in a f o r m a t r e a d y f o r d i g i t a l p r o c e s s i n g , w h i c h in t u r n p e r m i t s e x t r a c t i o n o f b i o logically useful structural information from n o i s y images.
INTRAMEMBRANEOUS PARTICLES ASSOCIATED WITH S U R F A C E IMMUNOGLOBULIN RECEPTORS IN T H E P L A S M A M E M B R A N E O F B L Y M P H O C Y T E S C.
De G r o o t ,
W.
Leene
L a b o r a t o r y of H i s t o l o g y and Cell Biol o g y , U n i v e r s i t y of A m s t e r d a m , 1 e C o n e t . H u y g e n s s t r a a t 20, 1054 BW A m s t e r d a m , The N e t h e r l a n d s
C o n v i n c i n g e v i d e n c e is a c c u m u l a t i n g t h a t s u r f a c e i m m u n o g l o b u l i n (sIg) r e c e p tor m o l e c u l e s of B l y m p h o c y t e s p o s s e s s two " e x t r a " h y d r o p h o b i c a m i n o a c i d seq u e n c e s w h e n c o m p a r e d w i t h s e c r e t e d imm u n o g l o b u l i n s . I-3 These hydrophobic p e p t i d e s a r e b e l i e v e d to f o r m a t r a n s m e m b r a n e s t r u c t u r e t h a t p l a y s an i m p o r t a n t r o l e in t h e t r a n s f e r o f a s i g n a l to the c y t o p l a s m . In t h e p r e s e n t s t u d y w e h a v e t r i e d b y f r e e z e - f r a c t u r i n g to v i s u a l i z e i n t r a m e m b r a n e o u s p a r t i c l e s (IMPs) s p e c i f i c a l l y a s s o c i a t e d w i t h sIg r e c e p t o r m o l e c u l e s . s I g r e c e p t o r s .on m o u s e a n d r a b b i t s p l e e n B l y m p h o c y t e s w e r e r e d i s t r i b u t e d by a n t i b o d i e s i n t o p a t c h e s and g l u t a r a l d e h y d e f i x e d c e l l s f r o z e n in 35% g l y c e r o l w e r e