Colloidal Gold Immunoreplica Method

12 C o l l o i d a l I m m u n o r e p l i c a

Μ .

V .

N E R M U T

a n d

G o l d M e t h o d

A .

N I C O L

National Institute for Medical Research The Ridgeway, M i l l H i l l London, NW7, United Kingdom

INTRODUCTION IMMUNOREPLICA METHOD Preparation o f Experimental Material COLLOIDAL G O L D IMMUNOLABELING Use o f Immunofluorescence to Develop Labeling Protocol Antibodies Immunogold Conjugates Controls Basic Labeling Protocol PROCESSING F O R ELECTRON MICROSCOPY Critical-Point Drying Freeze-Drying Comparison between Critical-Point Drying and Freeze-Drying Cleaning o f Replicas Whole Mounts MICROSCOPY AND INTERPRETATION CONCLUDING R E M A R K S REFERENCES

349 Colloidal Gold: Principles, Methods, and Applications, Vol. 1

Copyright © 1989 by Academic Press, Inc. Allrightsof reproduction in any form reserved.

Μ . V . Nermut and A . Nicol

350 INTRODUCTION

I m m u n o l a b e l i n g o f cell c o n s t i t u e n t s using fluorescent probes a n d light m i c r o s c o p y is n o t always a straightforward enterprise. I m m u n o l a b e l i n g using colloidal gold-conjugated p r o b e s a n d e l e c t r o n m i c r o s c o p y is m u c h less so. T h e p r i m a r y reason for this is t h e r e q u i r e m e n t for adequate ultrastruc­ tural preservation t o m a k e o b s e r v a t i o n at high magnifications sensible. T h e necessary c h e m i c a l fixation t e n d s t o inactivate t o s o m e degree antigenic d e t e r m i n a n t s a n d also tends t o prevent ready access o f i m m u n o r e a g e n t s , particularly colloidal gold conjugates, t o t h e antigens. D e p e n d i n g o n t h e location a n d properties o f antigens within t h e cell, m a n y t e c h n i q u e s for their ultrastructural i m m u n o l o c a l i z a t i o n h a v e b e e n devised. T h e i m m u n o r e p l i c a m e t h o d described herein largely c i r c u m v e n t s t h e p r o b l e m o f i m m u n o r e a gent access as t h e antigens o f interest are e x p o s e d o n t h e cell surface t o b e replicated. Although particularly suited t o t h e study o f antigens present o n t h e p r o t o p l a s m i c surface o f s u b s t r a t u m adherent m e m b r a n e s o f cultured cells, the i m m u n o r e p l i c a m e t h o d c a n b e applied t o the p r o t o p l a s m i c a n d external m e m b r a n e surfaces o f b o t h a t t a c h e d a n d suspension cultured cells. It is also applicable t o the study o f a n y small biological structure (cell organ­ elles, viruses, a n d m a c r o m o l e c u l e s ) adsorbed o n t o a charged surface such as a n alcian b l u e - c o a t e d coverslip. W h a t are t h e advantages a n d l i m i t a t i o n s o f surface replicas i n c o m p a r i s o n with ultrathin sections? All p l a s m a m e m b r a n e surfaces c a n b e visualized in ultrathin sections, b u t o n l y in cross s e c t i o n s a n d with t h e relatively low resolution o f positive staining as p r o d u c e d b y r o u t i n e processing ( F i g . 1). Serial sections followed b y computer-assisted r e c o n s t r u c t i o n c a n give at least s o m e i n f o r m a t i o n a b o u t the distribution o f structural e l e m e n t s o n b o t h t h e p r o t o p l a s m i c a n d e x t e r n a l surfaces. Surface replicas, o n t h e o t h e r h a n d , provide a n en face view o f t h e o u t e r o r i n n e r p l a s m a m e m b r a n e surfaces, a n d c o m b i n e d with g o o d structural preservation a n d high-resolution shadowing they represent a great step forward in o u r studies o f cell surfaces. B o t h m e m b r a n e - a s s o c i a t e d protein particles a n d cytoskeletal filaments c a n b e visualized with u n p r e c e d e n t e d clarity a n d detail (e.g., H e u s e r a n d E v a n s , 1 9 8 0 ; Aggeler et al, B o h n et al,

1 9 8 3 ; N e r m u t et al., 1 9 8 6 ; N i c o l a n d N e r m u t , 1 9 8 7 ;

1 9 8 7 ) . N e w procedures for t h e visualization o f t h e o u t e r a n d

i n n e r p l a s m a m e m b r a n e surfaces developed since t h e late 1 9 7 0 s have super­ seded the early a t t e m p t s at exposing cell surfaces b y freeze-fracture deep etching as used b y several authors, usually in c o m b i n a t i o n with ferritin i m m u n o l a b e l i n g ( P i n t o da Silva et al,

1 9 7 1 ; P i n t o da Silva a n d N i c o l s o n ,

1 9 7 4 ) o r gold labeling (Severs a n d R o b e n e k , 1 9 8 3 ) . T h e l i m i t a t i o n o f deep etching is that only a small surface area o f t h e cell is exposed. I n this paper we shall deal m a i n l y with t h e preparation a n d gold i m m u n o l a b e l i n g o f o u t e r a n d i n n e r cell surfaces.

351

Colloidal Gold Immunoreplica M e t h o d

Fig. 1. A conventional thin section through a cultured fibroblast cut at a right angle to the substratum. DM, dorsal membrane; VM, ventral membrane; FC, focal contacts (areas of tight interaction of the cell with the stubstratum). 1 0 , 0 0 0 X .

IMMUNOREPLICA

METHOD

Preparation of Experimental Cultured

Cell

Material

Monolayers

(a) Outer surface of "dorsal"

membrane

o f substratum-attached cultured

cells. T h e cells are plated o n t o a suitable substratum, allowed t o a t t a c h a n d spread as required, a n d t h e n rinsed with phosphate-buffered saline ( P B S ) prior t o fixation, labeling, drying a n d shadowing. R e p l i c a t i o n o f cell m o n o ­ layers is usually without serious p r o b l e m s , a n d several applications have b e e n published (e.g., Peters, 1 9 7 7 ; S c h w a r z et al,

1 9 7 6 ) . E x a m p l e s o f gold

i m m u n o l a b e l i n g include transferrin receptors ( H o p k i n s , 1 9 8 5 ) , L D L recep­ tors ( R o b e n e k et al, 1 9 8 2 , 1 9 8 3 ) , a n d virus-infected cells ( M a n n w e i l e r et al, 1 9 8 2 , 1 9 8 7 ) . Figure 2 shows gold i m m u n o l a b e l i n g o f outer surface o f a c h i c k e m b r y o ( b ) Outer surface of "ventral'

fibronectin

on the

fibroblast. membranes

o f a t t a c h e d cultured cells. R e ­

versing a cell m o n o l a y e r is rather difficult t o a c h i e v e with fibroblastic cells ( R e v e l a n d W o l k e n , 1 9 7 3 ) ; m o r e success has b e e n o b t a i n e d with sheets o f epithelial cells ( M u l l e r a n d G i m b r o n e , 1 9 8 6 ) , b u t n o i m m u n o l a b e l i n g has b e e n reported until n o w . W e have developed a procedure for reversing m o n o l a y e r s o f fibroblastic cells ( N e r m u t a n d B u r t , 1 9 8 9 ) . Briefly, a c o n ­ fluent m o n o l a y e r is e m b e d d e d in gelatin at r o o m t e m p e r a t u r e , t h e n c o o l e d d o w n o n i c e , lifted, a n d reversed b y m e a n s o f a piece o f m i c a i n c o r p o r a t e d in t h e gelatin. T h i s is followed b y prefixation a n d gold i m m u n o l a b e l i n g (Fig. 3 ) . (c) Protoplasmic

surface

of "ventral*

membrane

o f substratum a t t a c h e d

352

Μ . V. Nermut and A. Nicol

Fig. 2. Immunoreplica of the external surface of the "dorsal" membrane of a chick fibro­ blast labeled for fibronectin using rabbit antibody followed by colloidal gold (15 nm in diame­ ter) conjugated to goat anti-rabbit antibody. The cells were prefixed with glutaraldehyde and postfixed with glutaraldehyde, O s 0 4, and uranyl acetate, and freeze-dried before unidirectional shadowing (40°) and replication. Note a few unshadowed "stray" gold particles (arrows) as well as numerous "protein" particles about 10 nm in diameter. 60,000 X.

353

Colloidal Gold Immunoreplica M e t h o d

cells. Several procedures have b e e n described for b r e a k i n g o p e n cells in m o n o l a y e r s a n d washing away t h e b u l k o f t h e cell c o n t e n t t o leave b e h i n d only substratum-attached

m e m b r a n e a n d / o r focal adhesions. T h e m o s t

c o m m o n a n d successful procedure is t h e lysis-squirting m e t h o d ( C l a r k e et αί,

1 9 7 5 ) . O t h e r o p t i o n s are b r i e f s o n i c a t i o n o r scraping with a wire l o o p

(Aggeler a n d W e b b , 1 9 8 2 ; N e r m u t , 1 9 8 2 , for references a n d description). T h e " s a n d w i c h " t e c h n i q u e described b e l o w c a n also serve t h e purpose. A slightly m o r e c o m p l i c a t e d procedure h a s b e e n described b y M a s o n a n d J a c o b s o n ( 1 9 8 5 ) , b u t n o gold-labeled replicas have b e e n described using their m e t h o d . T h e r e are a few i m p o r t a n t points t o m a k e as far as lysis-squirting is c o n ­ cerned. Before squirting, cells are usually e x p o s e d t o distilled water o r hypo­ t o n i c buffer, which causes lysis o r i n m o s t cultured cells o n l y swelling. A v n u r a n d G e i g e r ( 1 9 8 1 ) pointed o u t t h a t w h e n this is carried out at a lower p H such as 6 . 1 , cells flatten d o w n a n d m a n y close c o n t a c t s develop. T h i s is obviously suitable for o b t a i n i n g large pieces o f ventral m e m b r a n e s . H o w ­ ever, for studies o f isolated focal adhesions a p H o r 7 . 0 - 7 . 2 is preferred. Also, s o m e cells m a y require certain p r e t r e a t m e n t s that stabilize t h e p l a s m a m e m b r a n e a n d prevent gross defects. A v n u r a n d G e i g e r ( 1 9 8 1 ) have always used 1 mM z i n c chloride in t h e p r e t r e a t m e n t ; others r e c o m m e n d a low c o n c e n t r a t i o n o f t a n n i c a c i d ( R u t t e r et αί,

1985).

T h e m a n n e r o f squirting c a n also influence t h e results. W h e r e a s gentle squirting from a Pasteur pipette leaves b e h i n d larger m e m b r a n e fragments, strong squirting from a syringe c o u l d r e m o v e all ventral m e m b r a n e e x c e p t adhesions. Procedures vary i n practice, a n d obviously m u s t b e applied flexi­ bly depending m a i n l y o n t h e cell type a n d t h e required results. I n t h e n e x t paragraph we shall describe t h e lysis-squirting procedure as it has b e e n used in o u r laboratory with different types o f cultured cells. Cells are plated o u t o n t o a suitable s u b s t r a t u m (usually a glass coverslip) at a density sufficient t o p r o d u c e a near-confluent m o n o l a y e r overnight. T h e cultures are rinsed briefly ( ~ 10 sec) in a P I P E S buffer ( 2 0 mM 1 0 0 mMKC1,

5 m M M g C l 2 , 3 mMEGTA,

PIPES,

p H 6 . 1 ) a n d t h e n transferred t o

2 0 % P I P E S buffer for 4 m i n . T h e swollen/lysed cells are t h e n sheared away from their substratum-adherent

membranes by means o f a jet o f P I P E S

buffer applied using a syringe fitted with either a h y p o d e r m i c needle o r a specially c o n s t r u c t e d flat nozzle ( N e r m u t , 1 9 8 6 ; N i c o l a n d N e r m u t , 1 9 8 7 ) (Fig. 4 A ) . T h e substratum-attached m e m b r a n e s are t h e n i m m e r s e d in t h e appropriate fixative (see later). T h e lysis-squirting procedure has b e e n used with m i n o r modifications in studies o f focal adhesions ( B a d l e y et αί, 1 9 8 0 ; A v n u r a n d Geiger, 1 9 8 1 ) o r virus assembly ( B a c h i a n d B u e c h i , 1 9 8 1 ; O d e n wald et αί,

1986).

T h e "dry-cleaving" ( M e s l a n d et αί, 1 9 8 1 ) a n d " d r y - b l o w i n g " ( A r a k i and

Colloidal Gold I m m u n o r e p l i c a M e t h o d

A

355

ventral membranes

cell monolayer

b

a

c ν dorsal (apical) Ssfefw. membrane

Β

ventral (basal) membrane

b

a

c

Fig. 4. Membrane preparation techniques. (A) Lysis-squirting, showing the stages de­ scribed in the text, (a) Cells attached to a coverslip are incubated in hypotonic buffer, (b) The bulk of the cellular components are sheared away by a stream of buffer applied using a syringe fitted with a specially constructed nozzle, (c) The adherent ventral membranes are left behind on the coverslip. (B) The sandwich technique for the preparation of dorsal and ventral mem­ branes, (a) Cells attached to a coverslip are rinsed free o f culture medium, (b) A polylysinecoated coverslip is placed onto the dorsal surface of the cells and allowed to adhere, (c) The coverslips are separated, cleaving the cells apart in the plane of the coverslips and resulting in predominantly "dorsal" membrane on the polylysine coated coverslip and "ventral" mem­ brane on the original coverslip. Many more cytoplasmic components are left associated with the ventral membrane using this method. (From Nermut, 1986, with permission.) Ogawa, 1 9 8 6 ) t e c h n i q u e s h a v e a p o t e n t i a l use for m a k i n g shadowed replicas or pseudoreplicas o f ventral m e m b r a n e s , b u t t h e gold i m m u n o l a b e l i n g is l i m i t e d t o t h e o u t e r cell surface. I m m u n o l a b e l i n g carried o u t after dry-cleav­ ing results in p o o r structural preservation ( M e s l a n d a n d Spiele, 1 9 8 4 ) a n d until n o w n o a t t e m p t h a s b e e n r e p o r t e d t o label m e m b r a n e - a s s o c i a t e d p r o ­ teins at t h e e l e c t r o n m i c r o s c o p i c level. (d)

The protoplasmic

surface

of "dorsal

membrane"

c a n b e visualized b y

m e a n s o f t h e " s a n d w i c h " t e c h n i q u e ( F i g . 4 B ) , as described b y B a t t e n et al

Fig. 3 (opposite page). Outer surface of chick embryo fibroblasts ventral membrane labeled with polyclonal antibody to fibronectin receptor, kindly supplied by Dr. Κ. M. Yamada. Reversed monolayers were prefixed and gold immunolabeled using the "basic labeling proto­ col" (see text), critical-point dried, shadowed, and replicated. Stereo microscopy showed that the streaky gold-labeled areas are raised above the membrane, indicating that they are the focal contacts. 37,000 X .

357

Colloidal Gold Immunoreplica M e t h o d

( 1 9 8 0 ) , Aggeler a n d W e r b ( 1 9 8 2 ) , a n d R u t t e r et al. ( 1 9 8 5 ) . O n e c a n also, after a b r i e f t r e a t m e n t with E D T A , reverse t h e m o n o l a y e r o n t o a n alcian bluec o a t e d coverslip a n d t h e n squirt t h e cells over as described in ( c ) . G o l d i m m u n o l a b e l i n g c a n b e carried o u t after squirting ( F i g . 5 ) , o r alternatively the outer surfaces o f cells c a n b e gold-labeled before sandwiching i f correla­ tion o f the localization o f surface receptors with s u b m e m b r a n o u s structures such as viral nucleocapsids is desired ( R u t t e r et al, 1 9 8 5 ; H o h e n b e r g et al, 1 9 8 5 ; B o h n et al,

1 9 8 7 ) . R e c e n t l y , R u t t e r et al ( 1 9 8 8 ) have used different

sizes o f colloidal gold particles t o label the o u t e r a n d i n n e r surfaces o f H e L a cells infected with measles virus ( F i g . 6 ) . A n o t h e r a p p r o a c h for correlation o f external label with s u b m e m b r a n o u s structures is t h e dry-cleaving t e c h n i q u e , though o n l y a few applications have up t o n o w b e e n published a n d m o s t l y without shadowing ( R o o s et al, Cell

1 9 8 5 ; W i e g a n t et al,

1986).

Suspensions

(a) Outer surface o f suspension cells. W a s h e d cells are usually a t t a c h e d t o coverslips positively charged with polylysine o r alcian b l u e o r alternatively t o glutaraldehyde-coated coverslips (see N e r m u t , 1 9 8 2 , for references). S u c h cells are usually high in profile after freeze-drying o r critical-point drying a n d are often covered with microvilli. It is therefore r e c o m m e n d e d t o apply rotary c a r b o n i n g from 4 5 ° t o 6 0 ° instead o f t h e usual 9 0 ° t o o b t a i n a c o n t i n u o u s replica. Freeze-fracture followed b y deep etching c a n b e a useful alternative in s o m e cases ( P i n t o da Silva a n d N i c o l s o n , 1 9 7 4 ; Severs a n d Robenek, 1983). ( b ) Protoplasmic

surface

of the plasmalemma

o f suspension cells. T h e

m o s t practical procedure is "lysis-squirting." Cells m u s t b e thoroughly washed a n d t h e n applied t o t h e positively charged surface o f a prepared glass coverslip o r a piece o f m i c a [see F i s h e r , ( 1 9 7 8 ) a n d N e r m u t ( 1 9 8 2 , 1 9 8 4 ) for detailed description]. It has b e e n applied t o red b l o o d cells ( L a n g et al, 1 9 8 1 ) a n d H e L a cells ( N e r m u t , 1 9 8 4 ) . Alternatively, cells c a n b e a t t a c h e d c o v a ­ lently t o glutaraldehyde-coated

coverslips before squirting (Aplin

and

Hughes, 1 9 8 1 ; B u e c h i a n d B a c h i , 1 9 7 9 ) . D e e p etching has also b e e n applied

Fig. 5 (opposite, top). Replica of protoplasmic surface of dorsal (apical) membrane of a measles virus-infected HeLa cell. Virus antigens on outer cell surface were labeled with antimeasles serum and protein A-gold conjugate. Note correlation of label with linear structures, most probably virus nucleocapsids. For technical details see Rutter et al (1985). 70,000 X . Figure kindly supplied by Dr. G. Rutter. Fig. 6 (opposite, bottom). Example of double immunolabeling of viral antigens on outer and inner faces of measles virus-infected HeLa cells (Rutter et al, 1988). Viral antigens on outer surface were immunolabeled using 25-nm gold particles, and viral phosphoproteins on inner surface were labeled with 13-nm gold particles. 72,000 X . Figure kindly supplied by Dr. G. Rutter.

358

Μ . V . Nermut and A. Nicol

t o red b l o o d cells, b u t with limited success ( S h o t t o n et al.,,1978),

a n d n o gold

labeling has b e e n used up t o n o w . Organelles,

Viruses,

and

Macromolecular

Complexes

Subcellular structures c a n b e adsorbed t o clean, alcian blue-coated o r poly-L-lysine-coated coverslips o r m i c a . G o l d i m m u n o l a b e l i n g is carried out in a test t u b e prior t o adsorption, o r o n coverslips following t h e s a m e proto­ c o l as described for cells. L a r g e subcellular structures such as cytoskeletons c a n also b e replicated, b u t since rotary shadowing is usually necessary t h e additional layer o f c a r b o n will considerably increase t h e t h i c k n e s s o f t h e unless a two-stage c a r b o n i n g procedure is used ( C e n t o n z e et αί,

filaments

1 9 8 5 ) . T h i s should i m p r o v e t h e stability o f t h e replica a n d k e e p t h e film thickness down. W e prefer w h o l e - m o u n t preparations, w h i c h require only a very thin stabilizing layer o f c a r b o n . R e p l i c a t i o n is often unnecessary in t h e case o f small structures such as viruses, lipid vesicles, o r plant thylakoids ( J a y etal.,

1985).

COLLOIDAL GOLD

IMMUNOLABELING

I m m u n o l a b e l i n g at t h e s u b m i c r o s c o p i c level requires preservation o f anti­ genicity o f the target antigen, preservation o f ultrastructure, a n d provision o f access for the i m m u n o r e a g e n t s t o t h e target antigen. Inevitably t h e fulfill­ m e n t o f these r e q u i r e m e n t s in o n e p r o t o c o l will in m o s t cases b e a c o m p r o ­ mise. F u r t h e r , this c o m p r o m i s e will vary for different target antigens ( B e n ­ dayan et αί, 1 9 8 7 ) a n d even for different antibodies t o t h e s a m e antigen as a result o f diverse epitopic specificities. W i t h t h e i m m u n o r e p l i c a t e c h n i q u e t h e accessibility o f t h e target antigen is essentially i n h e r e n t t o t h e procedure as, t o b e represented in the replica, t h e antigen m u s t b e present o n the prepared surface o f interest. T h u s , as there is n o n e e d for penetration agents such as detergent, m e m b r a n e s a n d m e m b r a n e - a s s o c i a t e d structures are well preserved. Preservation o f ultrastructure requires, i n m o s t cases, c h e m i c a l

fixation

before i m m u n o l a b e l i n g . S u c h prefixation should stabilize t h e ultrastructure sufficiently t o withstand t h e i m m u n o l a b e l i n g procedures b u t should b e " l i g h t " enough n o t t o excessively inactivate t h e target antigen. S o m e anti­ gens are highly sensitive t o fixation; others are a l m o s t indestructible. O t h e r factors that influence t h e o u t c o m e o f pre-fixation are c h e m i c a l n a t u r e o f fixative;

p H ; o s m o l a l i t y a n d n a t u r e o f fixation vehicle; a n d t e m p e r a t u r e ,

duration, a n d fixative c o n c e n t r a t i o n . T h e s e p r o b l e m s have b e e n extensively dealt with, a n d t h e reader is referred t o t h e relevant p u b l i c a t i o n s ( H a y a t , 1 9 8 1 , 1 9 8 6 ; P o l a k a n d V a r n d e l l , 1 9 8 4 ; B u l l o c k a n d Petrusz, 1 9 8 2 ; B u l l o c k ,

Colloidal Gold Immunoreplica M e t h o d

359

Fig. 7. Immunoreplica of N R K ventral membrane labeled for clathrin using a rabbit antibody (Nicol et al, 1987) followed by protein A-gold (17 nm). Prefixed with 1% glutaralde hyde for 10 min, postfixed, and processed by the critical-point drying protocol described. Unidirectional platinum/carbon shadowing (40°). The antibody was kindly supplied by Dr. Β. M. Jockusch. 37,000 X .

1 9 8 4 ; B e n d a y a n et al, 1 9 8 7 ; L e e n e n et al, 1 9 8 5 ) . T h e r e q u i r e m e n t for adequate ultrastructural preservation, however, effectively limits the c h o i c e o f prefixative t o formaldehyde a n d glutaraldehyde, although o t h e r crosslinking reagents such as the c a r b o d i i m i d e s have b e e n used with s o m e success (see Hayat, 1 9 8 1 ) . Glutaraldehyde gives superior preservation (Figs. 7 a n d 8 )

360

Μ . V . N e r m u t a n d A. N i c o l

361

Colloidal Gold Immunoreplica M e t h o d

b u t tends t o inactivate relatively m o r e antigens t h a n does formaldehyde o r t o prevent access due t o extensive cross-linking o f intracellular proteins ( W i l l i n g h a m a n d Paston, 1 9 8 5 ) . G l u t a r a l d e h y d e is thus t h e best c h o i c e i f the target antigen is n o t excessively inactivated. Several m e t h o d s have b e e n developed t o prevent o r m i n i m i z e i n a c t i v a t i o n o f antigenicity; for e x a m p l e , T o k u y a s u a n d S i n g e r ( 1 9 7 6 ) a n d T o k u y a s u ( 1 9 8 4 ) h a v e used ethylacetimidate to b l o c k a m i n o groups o n the antigen, restricting t h e degree o f sub­ sequent glutaraldehyde cross-linking. A n o t h e r a p p r o a c h is t o use a low c o n c e n t r a t i o n o f glutaraldehyde

i n s e q u e n c e o r in c o m b i n a t i o n

with

formaldehyde, for e x a m p l e , 3 % formaldehyde plus 0 . 1 % glutaraldehyde. W h e n glutaraldehyde is used as a prefixative, residual aldehyde groups that c a n c o n t r i b u t e t o b a c k g r o u n d b y binding antibodies should b e reduced using s o d i u m borohydride ( W e b e r et al., 1 9 7 8 ) , e t h a n o l a m i n e , glycine, o r lysine (Willingham, 1983; Willingham and Pastan, 1985). W h e n cells are prefixed with low c o n c e n t r a t i o n o f formaldehyde, long i m m u n o l a b e l i n g procedures c a n often result in t h e loss (washing o u t ) o f formaldehyde a n d d a m a g e t o t h e p l a s m a m e m b r a n e before postfixation c a n b e carried out. T h e reason is t h a t formaldehyde

fixation

is reversible, as

shown o n red b l o o d cells ( T o k u y a s u , 1 9 8 4 ) . A s prefixation is generally light, t h e preparation m u s t b e postfixed i m m e ­ diately after i m m u n o l a b e l i n g t o stabilize the structures during further pro­ cessing, such a s freeze-drying a n d critical-point drying. T h e r e q u i r e m e n t s o f postfixation are simply t h e m a x i m u m preservation o f ultrastructure (see processing for electron m i c r o s c o p y ) . Cells prefixed with formaldehyde c a n suffer considerably during i m m u n o l a b e l i n g , a n d such d a m a g e c a n n o t b e reversed b y postfixation. T o preserve p l a s m a m e m b r a n e integrity, it c a n b e worth trying t o fix cells with a low c o n c e n t r a t i o n o f glutaraldehyde o r t a n n i c acid after i n c u b a t i o n with t h e first a n t i b o d y .

Use of Immunofluorescence to Develop Labeling Protocol In m o s t cases where colloidal gold i m m u n o r e p l i c a labeling is considered, the overall distribution o f target antigen will already have b e e n well c h a r a c ­ terized using i m m u n o f l u o r e s c e n c e light m i c r o s c o p y . T h e c o n d i t i o n s o f la­ beling, fixation, a n t i b o d y dilutions, etc. provide useful i n f o r m a t i o n . I f the

Fig. 8 (opposite page). Immunoreplica of chick embryo fibroblast ventral membrane la­ beled for vinculin using an ascites mouse monoclonal antibody (courtesy of Dr. B. Geiger), a rabbit anti-mouse bridging antibody, then a colloidal gold (10 nm diameter) goat anti-rabbit conjugate. Vinculin is associated with areas of focal contacts. Prefixation was with 3% parafor­ maldehyde and the preparation was freeze-dried and shadowed prior to replication. Arrows point to unshadowed "stray" gold particles. 40,000 X.

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fixative used is already a n aldehyde, t h e n t h e labeling p r o t o c o l c o u l d directly b e applied t o gold i m m u n o l a b e l i n g . I f not, t h e n t h e fixative i n use should b e substituted with 3 % formaldehyde a n d t h e i m m u n o f l u o r e s c e n c e repeated. In short, we r e c o m m e n d t h e m a x i m u m use o f i m m u n o f l u o r e s c e n c e labeling t o help characterize appropriate fixation a n d a n t i b o d y labeling c o n d i t i o n s . R e f i n e m e n t s m a y b e m a d e a c c o r d i n g t o t h e results o b t a i n e d . O w i n g t o t h e autofluorescence i n d u c e d in m a n y biological materials b y glutaraldehyde fixation, it is generally best t o assess t h e effects o f glutaraldehyde fixation o n t h e target antigen using t h e full i m m u n o g o l d procedure rather t h a n b y i m ­ munofluorescence. If, however, t h e target antigen c a n n o t b e readily visualized b y i m m u n o f l u ­ orescence, t h e n c h a r a c t e r i z a t i o n o f appropriate labeling c o n d i t i o n s a n d dis­ c r i m i n a t i o n o f true labeling from b a c k g r o u n d will b e e x t r e m e l y difficult. I n these cases the use o f artificial test substrata such as t h e purified target antigen infiltrated i n t o a glutaraldehyde p o l y m e r i z e d b o v i n e s e r u m a l b u m i n b l o c k ( G a g n e a n d Miller, 1 9 8 7 ) o r i m m u n o b l o t t i n g m a y b e useful, b u t it m u s t b e r e m e m b e r e d that t h e target antigen test e n v i r o n m e n t will m o s t probably b e quite u n l i k e that found in

situ.

Antibodies A n t i b o d y preparations suitable for o t h e r colloidal gold i m m u n o l a b e l i n g m e t h o d s are equally suitable for i m m u n o r e p l i c a studies. G e n e r a l l y , the higher the purity o f t h e a n t i b o d y preparation, t h e less difficulty there is with b a c k g r o u n d labeling a n d o t h e r c o n t a m i n a t i n g affinities. High-titer antisera c a n s o m e t i m e s b e used at high dilutions where c o n t a m i n a t i n g antibodies are effectively diluted out. Unpurified antisera c a n b e used with confidence with t h e G L A D (gold-labeled antigen d e t e c t i o n ) m e t h o d ( L a r s s o n , 1 9 8 4 ) . T h e disadvantage o f t h e G L A D m e t h o d is that a specific gold-target antigen conjugate m u s t b e prepared in e a c h case. V e r y high dilutions o f a n t i b o d y in c o m b i n a t i o n with long i n c u b a t i o n t i m e s are unsuitable for m o s t i m m u n o r e ­ plica applications, as t h e lightly prefixed structure deteriorates o n prolonged i n c u b a t i o n . T h i s is especially n o t i c e a b l e w h e n formaldehyde fixation a l o n e is used.

Immunogold

Conjugates

A wide range o f particle sizes a n d c o n j u g a t e d a n t i b o d i e s are available. T h e m o s t useful sizes o f colloidal gold particles for i m m u n o r e p l i c a s are 5 , 1 0 , a n d 2 0 n m d i a m e t e r — 1 0 n m for general use, 5 n m for use where penetration a n d / o r high magnification observations are required, a n d 2 0 n m for a rela­ tively low magnification o r " s u r v e y " work. T h e y are e m p l o y e d in dilutions

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o f 1:1 to 1 : 1 0 o f that supplied, b u t routinely 1 : 5 is used in o u r laboratory. Provided correct washing o f the labeled preparation is observed, such dense suspensions d o n o t give rise t o high b a c k g r o u n d . P r o t e i n A - g o l d conjugates can b e used successfully ( N i c o l et al, 1 9 8 7 ) , b u t s o m e m o n o c l o n a l antibod­ ies do n o t b i n d protein A , a n d in o u r h a n d s such conjugates are less reliable t h a n a n t i b o d y - g o l d conjugates.

Controls T h e controls o f labeling specificity used with o t h e r i m m u n o l a b e l i n g tech­ niques are equally relevant here. T h e r e are three general categories o f nega­ tive controls. T h e s e are o m i s s i o n , substitution, a n d b l o c k i n g o f the specific antibodies. O m i s s i o n o f the specific a n t i b o d y is the simplest, a n d its i m p o r ­ t a n c e in regard t o i m m u n o g o l d labeling has b e e n recently highlighted ( B i r ­ rell et al., 1 9 8 7 ) . I n substitution the nature o f the specific antibody prepara­ tion is the i m p o r t a n t consideration. P o l y c l o n a l antisera should b e substituted with p r e i m m u n e s e r u m at t h e s a m e dilution. However, it is m o r e difficult t o find appropriate substitutions for specific a n t i b o d y preparations, for e x a m p l e , affinity-purified antibodies. S i m i l a r l y purified antibodies from the s a m e a n i m a l species b u t directed against a n irrelevant antigen m a y b e used. T h e s a m e considerations apply t o m o n o c l o n a l antibodies, where equivalent preparations (ascitic fluid, culture supernatant, o r a m o n o c l o n a l antibody t o a n unrelated antigen) c a n b e substituted. B l o c k i n g the labeling b y adding excess purified antigen t o the a n t i b o d y solution prior t o the labeling i n c u b a t i o n is a valuable c o n t r o l b u t is fre­ quently i m p r a c t i c a l due t o the l a c k o f purified antigen. A potential artifact that m a y o c c u r w h e n labeling ventral m e m b r a n e s prepared by lysis-squirting is the adsorption t o the substratum a n d subse­ q u e n t labeling o f soluble proteins released during squirting. However, in o u r experience, this is a rare event, j u d g i n g from the a b s e n c e o f ribosomes o n the m e m b r a n e s o r o n t h e substratum.

Basic Labeling Protocol • Prefixation: M a t e r i a l such as " v e n t r a l " m e m b r a n e s should b e fixed i m ­ mediately o n preparation with a n aldehyde such as 3 % paraformaldehyde in P B S for 15 m i n . • W a s h e s : two changes o f P B S followed b y o n e c h a n g e T B S / B S A ( 0 . 1 % ) . • P r i m a r y antibody: diluted in T B S / B S A ( 1 % ) for 3 0 m i n . • W a s h e s : three changes T B S / B S A ( 0 . 1 % ) for a total period o f 10 m i n . • I m m u n o g o l d reagent: diluted in T B S / B S A ( 1 % ) for 3 0 m i n .

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• W a s h e s : three c h a n g e s T B S / B S A ( 0 . 1 % ) for a total period o f 10 m i n a n d three changes P B S for a total period o f 10 m i n . • Postfixation a n d processing as described later. • N o t e s : P B S is D u l b e c c o ' s phosphate-buffered saline, with o r without c a l c i u m a n d m a g n e s i u m . T B S / B S A is Tris-buffered saline c o n t a i n i n g b o ­ vine serum a l b u m i n ( 2 0 m M T r i s , 1 5 0 m M N a C l , 0.1 % B S A , 2 0 m ¥ N a N 3 , p H 8 . 2 ; o r 2 0 m M T r i s , 1 5 0 m A f N a C l , 1 % B S A , 2 0 m M N a N 3 , p H 8 . 2 ) . All steps are carried o u t at r o o m t e m p e r a t u r e ( 2 0 ° C ) . W h e n using small vol­ u m e s o f i m m u n o r e a g e n t s it is necessary t o place the m a t e r i a l being labeled in a closed a n d / o r humidified c o n t a i n e r such as a small petri dish. T h e basic p r o t o c o l c a n easily a c c o m m o d a t e alternative labeling s c h e m e s such as t h e bridge t e c h n i q u e , i n w h i c h t h e p r i m a r y a n t i b o d y is followed b y a bridging antibody directed against t h e p r i m a r y antibody. T h e i m m u n o g o l d reagent t h e n is directed against the bridging a n t i b o d y . S u c h additional anti­ b o d y i n c u b a t i o n steps are followed b y washes as for t h e p r i m a r y a n t i b o d y step.

PROCESSING FOR ELECTRON MICROSCOPY T h e a i m o f this section is t o describe t h e processing m e t h o d s used a n d to i n d i c a t e usable variations, b u t for a n in-depth description o f t h e t e c h n i c a l aspects a n d m e t h o d o l o g i e s o f replication, critical-point drying, a n d freezedrying the reader is referred t o R o b a r d s a n d Sleytr ( 1 9 8 5 ) . T h e following processing is suitable for m e m b r a n e preparations.

Critical-Point Drying • Postfixation: 2 . 5 % glutaraldehyde in P B S ( p H 7 . 4 ) for 15 m i n ; three changes in P B S for a total period o f 10 m i n ; 2 % O s 0 4 in 0.1 Μ s o d i u m cacodylate buffer ( p H 7 . 4 ) for 15 m i n ; three changes in distilled water for a total period o f 5 m i n ; 1% uranyl a c e t a t e for 1 - 2 m i n ; rinse in distilled water. • D e h y d r a t i o n : 5 0 % e t h a n o l for 5 m i n ; 7 5 % e t h a n o l for 5 m i n ; three changes in 1 0 0 % e t h a n o l for 5 m i n e a c h . U s i n g a critical-point drying ( C P D ) apparatus, the e t h a n o l is replaced b y liquid C 0 2 . ( T h e use o f a c e t o n e before C P D is unnecessary for m o n o l a y e r s o f cells o r m e m b r a n e s a n d m a y prove damaging t o t h e m . ) T h e dried preparations are q u i c k l y transferred t o a v a c u u m c o a t i n g unit o r t o a d e s i c c a t o r for storage until replication. • R e p l i c a t i o n : Critical-point-drying preparations are usually replicated at r o o m temperature, a n d shadowing is d o n e from o n e direction at 4 0 ° o r at 2 0 - 3 0 ° for rotary shadowing. T h e c a r b o n support film is applied b y rotary deposition at 8 0 ° for m e m b r a n e preparations o r at 4 5 - 6 0 ° for w h o l e cells. • R e p l i c a release: T h e replicas are released (from glass coverslips) b y flota-

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tion o n dilute ( 4 % ) hydrofluoric acid a n d t h e n transferred over three changes o f distilled water t o t h e c l e a n i n g agent. R e p l i c a s from m i c a c a n b e floated directly o n bleach, b u t this c a n b e a slow process. It is advisable t o score the c a r b o n film first a n d leave the m i c a floating o n the b l e a c h for a few hours. T h e n the replica separates a n d t h e m i c a sinks after a gentle t o u c h with a pair o f tweezers. • R e p l i c a cleaning: B y floating o n c o n c e n t r a t e d b l e a c h ( N a - h y p o c h l o r i t e ) for 2 - 4 h r o r overnight o n a diluted b l e a c h . After cleaning, the replicas are floated o n three changes o f distilled water a n d then picked up o n t o T E M grids (thin-bar 4 0 0 - m e s h o r hexagonal grids are very suitable).

Freeze-Drying • Postfixation: 2 . 5 % glutaraldehyde in P B S ( p H 7 . 4 ) for 15 m i n t o 3 0 m i n ; three changes in P B S for a total period o f 10 m i n ; 2 % O s 0 4 in 0.1 Μ s o d i u m cacodylate buffer ( p H 7 . 4 ) for 15 m i n ; this step m a y b e omitted, but n o t i f whole m o u n t s are desired; three changes in distilled water for a total period o f 5 m i n ; 1% uranyl acetate for 1 - 2 m i n . • Freezing: Coverslips o r grids are washed repeatedly with distilled water, which should b e o f t h e highest available purity t o reduce the a m o u n t o f eutectic deposits left after drying. E x c e s s water is carefully b u t quickly drained from the reverse side o f t h e coverslip a n d from t h e e x p e r i m e n t a l face by touching a piece o f filter paper. T h e preparation is t h e n quickly plunged i n t o liquid nitrogen a n d m o u n t e d o n t o a freeze-drying s p e c i m e n stage precooled in the trough o f liquid nitrogen. W e use Balzers's " s p e c i m e n table for three s p e c i m e n s " modified t o h o l d coverslips instead o f grids. • Freeze-drying: W e use B a k e r s ' B A F 3 0 0 freeze-etch plant equipped with a counterflow loading system. T h e s p e c i m e n s are dried routinely at — 8 0 ° C 6 ( 1 9 3 K ) a n d in a v a c u u m better t h a n 5 X 1 0 ~ m b a r for 1 5 - 4 5 m i n (see N e r m u t , 1 9 7 7 , for details), with the actual t i m e depending o n the visually j u d g e d end point. • R e p l i c a t i o n : Shadowing a n d c a r b o n replication are carried out at the low temperature o f t h e s p e c i m e n stage. T h e w h o l e - m o u n t preparations are warmed up t o r o o m temperature before being r e m o v e d from the c h a m b e r . R e p l i c a release a n d cleaning are carried out as for critical-point drying. Comparison between Critical-Point Drying a n d FreezeDrying T h e relative merits o f these dehydration procedures have b e e n discussed by R o b a r d s a n d Sleytr ( 1 9 8 5 ) . Aggeler et al ( 1 9 8 3 ) observed a n excellent preservation o f b o t h the m e m b r a n e s a n d the cytoskeleton in glutaraldehyde-fixed a n d freeze-dried m a c r o p h a g e s . In o u r h a n d s freeze-drying is the

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superior m e t h o d for m e m b r a n e replicas. It gives g o o d definition o f finestructural features such as protein particles o n m e m b r a n e s , a n d is usually gentle t o m e m b r a n e s . However, it is necessary t o follow t h e original c o n d i ­ tions for drying ( N e r m u t et αϊ, 1 9 7 2 ) t o prevent over-drying, which results in collapse o f soft structures, c r a c k s in m e m b r a n e s , b r e a k s o f

filaments

or

cellular processes, a n d general flattening. T h i s c a n h a p p e n in particular i f drying is carried o u r at between - 5 0 ° C ( 2 2 3 K ) a n d - 3 0 ° C ( 2 4 3 K ) , as used for e x a m p l e b y K i s t l e r a n d K e l l e n b e r g e r ( 1 9 7 7 ) o r B u e c h i a n d B a c h i ( 1 9 7 9 ) . After i m m u n o l a b e l i n g , w h i c h c a n t a k e 2 - 3 hr, it is advisable t o postfix m e m b r a n e preparations with glutaraldehyde followed b y O s 0 4 a n d in s o m e cases with 0 . 2 % t a n n i c a c i d ( I s o b e a n d S h i m a d a , 1 9 8 6 ) . Freeze-drying re­ quires skill a n d is usually m o r e t i m e - c o n s u m i n g , as with m o s t e q u i p m e n t it is difficult t o process m o r e t h a n t w o preparations at a t i m e . U p t o 10 preparations c a n b e processed at a t i m e b y critical-point drying, a n d t h e procedure requires less dexterity. It is g o o d for preservation o f the three-dimensional structure, b u t m e m b r a n e s c a n develop holes a n d

fine

details are s o m e t i m e s obscured. Aggeler et al. ( 1 9 8 3 ) r e c o m m e n d fixing m e m b r a n e preparations with aldehydes followed b y lysine, O s 0 4 , t a n n i c acid, a n d uranyl acetate. T h u s , critical-point drying is useful for surveying multipreparation e x p e r i m e n t s , a n d freeze-drying is useful w h e n t h e best possible ultrastructural preservation is desired. Shadowing

is carried o u t best with P t / C using an e l e c t r o n - b e a m evapora­

t o r a n d a quartz crystal m o n i t o r . U n i d i r e c t i o n a l shadowing at a n angle o f 3 0 - 4 5 ° provides b o t h g o o d definition o f small structural features a n d in­ stant i n f o r m a t i o n a b o u t the t h r e e - d i m e n s i o n a l situation in the preparation. G o l d particles are usually distinctly shadowed, w h i c h helps t o discriminate between true surface labeling a n d stray gold particles ( F i g s . 2 , 7, a n d 8 ) . R o t a r y shadowing from 2 0 t o 3 0 ° proves superior for

filamentous

prepa­

rations such as t h e cytoskeleton, b u t is less useful for studies o f small particles a n d does n o t provide i n f o r m a t i o n a b o u t t h e height o f structures unless stereo pairs o f micrographs are t a k e n . T h e size o f particles including colloidal gold is considerably increased. R e p l i c a t i o n with c a r b o n c a n b e d o n e in t h e usual way, b u t whole cells o r cytoskeletons should b e replicated from 6 0 ° o r even 4 5 ° with t h e s p e c i m e n stage rotating during evaporation. S u c h replicas are m o r e resistant t o b r e a k ­ ing up during cleaning.

Cleaning o f Replicas W h e n floated off its support, the i m m u n o r e p l i c a is still sandwiched with t h e underlying fixed m a t e r i a l t o w h i c h t h e colloidal gold label was attached. G o l d particles o n the surface o f t h e fixed m a t e r i a l are trapped in t h e replica

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a n d retained when the fixed material a n d u n t r a p p e d gold particles b e l o w the surface are r e m o v e d b y t h e c l e a n i n g agents. U n s h a d o w e d " s t r a y " gold parti­ cles m a y m e a n that the replica was n o t c l e a n e d properly o r that s o m e gold particles were dislodged during t h e c l e a n i n g process. N o t all standard replica cleaning agents c a n b e used. B o t h chrome-sulfuric acid a l o n e a n d c o n c e n ­ trated bleach followed b y 6 0 % sulfuric a c i d c a n remove even t h e " t r a p p e d " gold particles. W e find 2 - 4 h r o n c o n c e n t r a t e d b l e a c h o r longer t r e a t m e n t s with dilute bleach the m o s t satisfactory for retention o f gold particles within the i m m u n o r e p l i c a . I n s o m e cases replicas are floated o n distilled water t o keep m e m b r a n e s a n d gold label associated with t h e replica ( R u t t e r et al, 1 9 8 8 ; K a n a n d P i n t o d a Silva, V o l u m e 2 , this series).

Whole Mounts I f the original preparation is m o u n t e d o n a n e l e c t r o n m i c r o s c o p e grid (e.g., cells cultured o n a F o r m v a r / c a r b o n - c o a t e d gold grid), t h e n the s a m e i m ­ m u n o r e p l i c a p r o t o c o l c a n b e used t o prepare whole m o u n t s (cells, m e m ­ branes, a n d adsorbed particles). T h e o n l y c h a n g e is t h a t after dehydration t h e preparation is left unshadowed o r m a y b e lightly shadowed. A l s o , t h e prepa­ ration m a y require stabilization b y a light rotary deposition o f c a r b o n . S u c h whole m o u n t s ( F i g . 9 ) are potentially valuable, giving i n f o r m a t i o n a b o u t the l o c a t i o n o f subsurface antigens, providing that such antigens are accessible t o the i m m u n o l a b e l i n g reagents.

MICROSCOPY

AND

INTERPRETATION

M o s t true labeling ( a n d true b a c k g r o u n d ) colloidal gold particles found in replicas are shadowed. T h i s is m o s t easily seen w h e n unidirectional shadow­ ing has b e e n used a n d t h e structure replicated is relatively flat a n d u n c o m ­ plicated. G o l d particles lacking shadow should b e observed with c a u t i o n as they m a y b e " s t r a y " rather t h a n " t r u e " particles, that is, particles displaced from their true l o c a t i o n o r deriving from labeled subsurface m a t e r i a l n o t represented i n t h e replica. S t e r e o p h o t o m i c r o g r a p h s prove very useful i n assisting j u d g e m e n t o f t h e validity o f given particles, a n d this is especially t h e case for whole m o u n t s where " s t r a y " gold particles c a n often a t t a c h t o t h e reverse side o f the support film ( F i g . 9 ) . Q u a n t i t a t i o n o f particle distribution within a replica m a y b e useful as a n aid t o distinguishing low levels o f labeling from b a c k g r o u n d . S u c h q u a n t i t a ­ t i o n should b e used with c a u t i o n , as different surfaces represented within the replica (glass, m i c a , c a r b o n film, a n d extracellular m a t r i x ) n e e d n o t attract t h e s a m e b a c k g r o u n d levels. I n general, labeling in i m m u n o r e p l i c a c a n n o t

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b e considered quantitative, although there m a y b e s o m e specialized situa­ t i o n s in which q u a n t i t a t i o n c o u l d b e envisaged, such as dilute m i x t u r e s o f m a c r o m o l e c u l e s adsorbed o n t o a c l e a n surface.

CONCLUDING

R E M A R K S

T h e i m m u n o r e p l i c a t e c h n i q u e is t h e m e t h o d o f c h o i c e for studies o f b o t h external a n d internal cell surface antigens, their topography a n d b e h a v i o r during t h e cell cycle, cell l o c o m o t i o n o r t r a n s f o r m a t i o n . It provides high resolution o f b o t h the labeling a n d the m e m b r a n e - a s s o c i a t e d structures. S m a l l distinct particles h a v e b e e n visualized o n b o t h t h e o u t e r a n d i n n e r p l a s m a m e m b r a n e surfaces b y freeze-dry replication ( F i g . 2 a n d 7 ) , m o s t probably protein m a c r o m o l e c u l e s (e.g., cellular receptors). However, high resolution is at v a r i a n c e with a n overall localization o f surface antigens at low magnification. N o t only are

fine-structural

details

lost, b u t colloidal gold o f a b o u t 2 0 n m is needed t o b e visible at low magnifi­ cation, which reduces resolution a n d yield. High-resolution s c a n n i n g elec­ t r o n m i c r o s c o p y is t h e o b v i o u s solution here. T h e reversibility o f formaldehyde fixation c a n b e a n o t h e r p r o b l e m . S i n c e t h e initial c o n c e n t r a t i o n is rather low, t h e structural preservation is n o t o f a high standard in the first place. F u r t h e r m o r e , t h e loss o f formaldehyde from the cells during labeling c a n result in t h e deterioration o f ultrastructure. T h i s has b e c o m e evident in o u r studies o f cell surface receptors, w h e n defects have b e e n observed in dorsal m e m b r a n e after either C P D o r F D . In such cases it was therefore necessary t o i n t r o d u c e a n i n t e r m e d i a t e fixation t o i m p r o v e t h e structural preservation. A l c o h o l i c dehydration before C P D c a n also c o n t r i b ­ ute t o s o m e m e m b r a n e defects. W h e n cells are grown a n d labeled o n grids, b o t h surfaces are usually m a r k e d with gold particles. S t e r e o pairs o f m i c r o g r a p h s help t o d i s c r i m i n a t e between dorsal a n d ventral m e m b r a n e labeling. Alternatively, light shadow­ ing ( b e t t e r from 3 0 ° t h a n 4 5 ° ) m a k e s it evident which particles are under-

Fig. 9 (oppositepage). Whole-mount preparation of N R K "ventral" membrane labeled for vinculin using a commercially available mouse monoclonal antibody (Bio-Yeda, Rehovot, Israel) followed by rabbit anti-mouse bridging antibody and finally goat anti-rabbit conjugated colloidal gold (10 nm). The membranes were prefixed with 3% paraformaldehyde for 10 min, postfixed, and dehydrated using the critical-point drying protocol before being lightly shadowed (40°) with platinum - carbon and stabilized by rotary carbon deposition (80°). When viewed in stereo, some gold particles can be identified on the undersurface of the Formvar support film (arrows), whereas the remainder truly indicate the presence of vinculin in the microfilament bundle terminus. Total tilt angle is 12°. 7 5 , 0 0 0 X .

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n e a t h the cell, b e c a u s e t h e y will n o t b e shadowed. T h i s is, however, l i m i t e d t o flat cells such as

fibroblasts.

D e t e r g e n t - e x t r a c t e d cells c a n also b e replicated, b u t t h e o r g a n i c m a t e r i a l is usually o n l y partly r e m o v e d o n c l e a n i n g (resulting in a pseudoreplica), a n d b o t h rotary shadowing a n d c a r b o n i n g i n c r e a s e c o n s i d e r a b l y t h e size o f t h e cytoskeletal e l e m e n t s a n d r e d u c e resolution. W h o l e - m o u n t p r e p a r a t i o n s are therefore preferred for studies o f p e r m e a b i l i z e d cells.

Our thanks are due to Philip Eason, Jim Burt, and Liz Hirst for technical assistance and to Marilyn Brennan for typing the manuscript.

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