- Email: [email protected]
Freeze-etch immuno-gold labeling of E. Coli outer membrane antigens
406
Abstracts of The Netherlands Society for Electron Microscopy & Royal Microscopical Socie~
far m a t r i x m a t e r i a l may cause a local increase in density, r e s u l t i n g in "specific", but false, localization. We have tried to c i r c u m v e n t this by using f r e e z e - f r a c t u r e d tissue and inv e s t i g a t e d the d i f f u s i o n p r o b l e m in b o v i n e s e r u m a l b u m i n gels, c r o s s - l i n k e d with g l u t a r a l d e h y d e , in w h i c h v a r y i n g amounts (i-i00 mM) of Ca or Na ions had been incorporated. Small b l o c k s w e r e q u i c k l y frozen in l i q u i d - n i t r o g e n c o o l e d propane, and b r o k e n with a prec h i l l e d scalpel. S u b s e q u e n t l y they were t r a n s f e r r e d to a p r e c h i l l e d r e a g e n t - f i x a t i v e m i x t u r e and a l l o w e d to thaw. D e h y d r a t i o n and e m b e d d i n g took p l a c e a c c o r d i n g to s t a n d a r d procedures. The d i s t r i b u t i o n of the p r e c i p i t a t e was uneven. A c r u s t of c r y s t a l l i n e m a t e r i a l was found on the fracture face, a ca 1 ~ w i d e area b e l o w showing little or no precipitate. D e e p e r in the gel the p r e c i p i t a t e was fine and distributed homogeneously. Varying amounts of Ca, Na and K were recorded. In all cases K gave a high signal. At high Ca c o n c e n t r a t i o n s the Ca peak was c l e a r l y d i s t i n c t from the n e i g h b o r i n g K peak and m i g h t be used for q u a n t i t a tive m e a s u r e m e n t s . WD X-ray analysis i n d i c a t e d that Ca tends to d i f f u s e to the f r a c t u r e face during thawing from the b o r d e r area and is m a i n l y found in the crust. Small pieces of P h o r m i a (Insecta, Diptera) flight m u s c l e showed a similar crust and sometimes a p r e c i p i t a t e - f r e e b o r d e r zone as well. C a l c i u m could be l o c a l i z e d in the m i t o c h o n d r i a and in the m y o f i b r i l s . A distinct mitochondrial l o c a l i z a t i o n was e s p e c i a l l y found in tissue fixed in K P A - g l u t a r a l d e h y d e and p o s t - f i x e d in OsO4 in Na c a c o d y l a t e buffer. In this case Ca may have been r e p l a c e d p a r t l y by Na. F r e e z e - f r a c t u r e d m a t e r i a l may be used to p r e v e n t false l o c a l i z a t i o n c a u s e d by d i f f u s i o n barriers, but does not p r o v i d e a s o l u t i o n to the d i f f u s i o n p r o b l e m itself.
FREEZE-ETCH IMMUNO-GOLD LABELING E. COLI O U T E R M E M B R A N E A N T I G E N S
OF
W. F. Voorhout, J. J. M. L e u n i s s e n Bijvelt*, J. L. M. L e u n i s s e n and A. J. V e r k l e i j Institute of Molecular Biology and *Department of Molecular Cell Biology, State University of Utrecht,
Padualaan
8,
3584
CH
Utrecht
PhoE 3, one of the E. coli outer membrane pore protein complexes, has been localized on u l t r a t h i n c r y o s e c t i o n s by i m m u n o - g o l d labeling. I Here we describe for the first time the l a b e l i n g of PhoE 3 in f r e e z e - e t c h replicas using m o n o c l o n a l a n t i b o d i e s and the immunogold method. E. coli cells were fixed and incubated with m o n o c l o n a l antibodies, rabbita n t i - m o u s e IgG and p r o t e i n A - g o l d (12 nm ~) according to s t a n d a r d procedures. A f t e r the final w a s h i n g the cells were frozen by jet-freezing, f r e e z e - f r a c t u r e d and etched. S p e c i f i c a l l y b o u n d gold p a r t i c l e s are easily d i s c e r n e d from a s p e c i f i c a l l y b o u n d p a r t i c l e s in freezeetch replicas, since only the first d i s p l a y the p r e s e n c e of shadow-cones. It was found that a p p r o x i m a t e l y 1% of the cells was labeled, w h i c h was confirmed by i m m u n o f l u o r e s c e n c e and w h o l e m o u n t techniques. However, this is not in a g r e e m e n t w i t h the results o b t a i n e d using c r y o - u l t r a m i c r o t o m y and immunolabeling, w h i c h r e s u l t e d in dense labeling of the outer m e m b r a n e of almost all s e c t i o n e d bacteria. An e x p l a n a t i o n may be that b i n d i n g of a n t i b o d y tQ the outer m e m b r a n e protein does not occur due to steric hindrance, since PhoE 3 forms c o m p l e x e s with l i p o p o l y s a c c h a r i d e in the p r e s e n c e of Ca 2+. This e x p l a n a t i o n is s u p p o r t e d by the o b s e r v a t i o n that labeled cells w h i c h were s e c t i o n e d and i n c u b a t e d again with a n t i b o d y and p r o b e showed labeling of a l m o s t all cells, w h i l e sections w h i c h w e r e not treated with a n t i b o d y only showed labeling of about 1% of the cells. i.
J. Leunissen, M. van Damme-Jongsten, J. Tommassen, B. Lugtenberg and A. J. Verkleij, Cell Biol. Int. Rep. 8 (1984) 187.
RAPID D I A G N O S I S OF HERPES VIRUS F I E L D ISOLATES WITH C O L L O I D A L GOLD IMMUNE E L E C T R O N MICROSCOPY~ I. D I F F E R E N T I A T I O N B E T W E E N H U M A N HERPES S I M P L E X V I R U S E S TYPE 1 AND TYPE 2 U S I N G M O N O C L O N A L A N T I B O D I E S . II. T Y P I N G OF "EQUINE HERPES VIRUS A B O R T I O N " Joh. Vreeswijk, F. W a g e n a c ~
P. de K r e e k
and
Central Veterinary Institute, ogy Department, P.O. Box 365, AJ Lelystad
Virol8200
Abstracts of The Netherlands Society for Electron Microscopy & Royal Microscopical Socie~
far m a t r i x m a t e r i a l may cause a local increase in density, r e s u l t i n g in "specific", but false, localization. We have tried to c i r c u m v e n t this by using f r e e z e - f r a c t u r e d tissue and inv e s t i g a t e d the d i f f u s i o n p r o b l e m in b o v i n e s e r u m a l b u m i n gels, c r o s s - l i n k e d with g l u t a r a l d e h y d e , in w h i c h v a r y i n g amounts (i-i00 mM) of Ca or Na ions had been incorporated. Small b l o c k s w e r e q u i c k l y frozen in l i q u i d - n i t r o g e n c o o l e d propane, and b r o k e n with a prec h i l l e d scalpel. S u b s e q u e n t l y they were t r a n s f e r r e d to a p r e c h i l l e d r e a g e n t - f i x a t i v e m i x t u r e and a l l o w e d to thaw. D e h y d r a t i o n and e m b e d d i n g took p l a c e a c c o r d i n g to s t a n d a r d procedures. The d i s t r i b u t i o n of the p r e c i p i t a t e was uneven. A c r u s t of c r y s t a l l i n e m a t e r i a l was found on the fracture face, a ca 1 ~ w i d e area b e l o w showing little or no precipitate. D e e p e r in the gel the p r e c i p i t a t e was fine and distributed homogeneously. Varying amounts of Ca, Na and K were recorded. In all cases K gave a high signal. At high Ca c o n c e n t r a t i o n s the Ca peak was c l e a r l y d i s t i n c t from the n e i g h b o r i n g K peak and m i g h t be used for q u a n t i t a tive m e a s u r e m e n t s . WD X-ray analysis i n d i c a t e d that Ca tends to d i f f u s e to the f r a c t u r e face during thawing from the b o r d e r area and is m a i n l y found in the crust. Small pieces of P h o r m i a (Insecta, Diptera) flight m u s c l e showed a similar crust and sometimes a p r e c i p i t a t e - f r e e b o r d e r zone as well. C a l c i u m could be l o c a l i z e d in the m i t o c h o n d r i a and in the m y o f i b r i l s . A distinct mitochondrial l o c a l i z a t i o n was e s p e c i a l l y found in tissue fixed in K P A - g l u t a r a l d e h y d e and p o s t - f i x e d in OsO4 in Na c a c o d y l a t e buffer. In this case Ca may have been r e p l a c e d p a r t l y by Na. F r e e z e - f r a c t u r e d m a t e r i a l may be used to p r e v e n t false l o c a l i z a t i o n c a u s e d by d i f f u s i o n barriers, but does not p r o v i d e a s o l u t i o n to the d i f f u s i o n p r o b l e m itself.
FREEZE-ETCH IMMUNO-GOLD LABELING E. COLI O U T E R M E M B R A N E A N T I G E N S
OF
W. F. Voorhout, J. J. M. L e u n i s s e n Bijvelt*, J. L. M. L e u n i s s e n and A. J. V e r k l e i j Institute of Molecular Biology and *Department of Molecular Cell Biology, State University of Utrecht,
Padualaan
8,
3584
CH
Utrecht
PhoE 3, one of the E. coli outer membrane pore protein complexes, has been localized on u l t r a t h i n c r y o s e c t i o n s by i m m u n o - g o l d labeling. I Here we describe for the first time the l a b e l i n g of PhoE 3 in f r e e z e - e t c h replicas using m o n o c l o n a l a n t i b o d i e s and the immunogold method. E. coli cells were fixed and incubated with m o n o c l o n a l antibodies, rabbita n t i - m o u s e IgG and p r o t e i n A - g o l d (12 nm ~) according to s t a n d a r d procedures. A f t e r the final w a s h i n g the cells were frozen by jet-freezing, f r e e z e - f r a c t u r e d and etched. S p e c i f i c a l l y b o u n d gold p a r t i c l e s are easily d i s c e r n e d from a s p e c i f i c a l l y b o u n d p a r t i c l e s in freezeetch replicas, since only the first d i s p l a y the p r e s e n c e of shadow-cones. It was found that a p p r o x i m a t e l y 1% of the cells was labeled, w h i c h was confirmed by i m m u n o f l u o r e s c e n c e and w h o l e m o u n t techniques. However, this is not in a g r e e m e n t w i t h the results o b t a i n e d using c r y o - u l t r a m i c r o t o m y and immunolabeling, w h i c h r e s u l t e d in dense labeling of the outer m e m b r a n e of almost all s e c t i o n e d bacteria. An e x p l a n a t i o n may be that b i n d i n g of a n t i b o d y tQ the outer m e m b r a n e protein does not occur due to steric hindrance, since PhoE 3 forms c o m p l e x e s with l i p o p o l y s a c c h a r i d e in the p r e s e n c e of Ca 2+. This e x p l a n a t i o n is s u p p o r t e d by the o b s e r v a t i o n that labeled cells w h i c h were s e c t i o n e d and i n c u b a t e d again with a n t i b o d y and p r o b e showed labeling of a l m o s t all cells, w h i l e sections w h i c h w e r e not treated with a n t i b o d y only showed labeling of about 1% of the cells. i.
J. Leunissen, M. van Damme-Jongsten, J. Tommassen, B. Lugtenberg and A. J. Verkleij, Cell Biol. Int. Rep. 8 (1984) 187.
RAPID D I A G N O S I S OF HERPES VIRUS F I E L D ISOLATES WITH C O L L O I D A L GOLD IMMUNE E L E C T R O N MICROSCOPY~ I. D I F F E R E N T I A T I O N B E T W E E N H U M A N HERPES S I M P L E X V I R U S E S TYPE 1 AND TYPE 2 U S I N G M O N O C L O N A L A N T I B O D I E S . II. T Y P I N G OF "EQUINE HERPES VIRUS A B O R T I O N " Joh. Vreeswijk, F. W a g e n a c ~
P. de K r e e k
and
Central Veterinary Institute, ogy Department, P.O. Box 365, AJ Lelystad
Virol8200