Effects of different fixative solutions on labeling of Concanavalin-A ...

Histochemistry(1981) 71:559-565

Histochemistry 9 Springer-Verlag 1981

Effects of Different Fixative Solutions on Labeling of Concanavalin-A Receptor Sites in Human T-Lymphocytes J. Renau-Piqueras ~*, E. Knecht 2, and J. Hernandez-Yago 1 t Instituto de InvestigacionesCitol6gicas, Amadeo de Saboya4, Valencia-10, Spain, and 2 Department of BiologicalSciences, Stanfor University, Stanfor, California94305, USA

Summary. We have studied the effect of several fixative solutions on the number of Concanavalin-A (Con-A) receptor sites of human peripheral blood T-lymphocytes. Cells treated with different fixative solutions (glutaraldehyde (G) ; formaldehyde (F) ; G + F; osmium tetroxide (Os) ; Os + G; Os + F; and Os + G + F) were labeled with a Con-A gold labeled horseradish peroxidase (HRP) complex and the number of gold particles on the lymphocytic surface was evaluated. Comparison of cells treated with the different fixatives used showed significant differences in the density of labeling. After G fixation the number of gold particles was lower than after fixation with Os or F. Moreover, G used in combination with F or Os reduced the labeling obtained when the two latter fixatives were used alone.

Introduction The surface coat is responsible for a wide variety of biological functions and properties of the cells (Luft 1976 ; Bona 1975 ; Santer 1977). Therefore, evaluation of their components is very important for understanding many of these functions and several cytochemical methods have been used to determine the nature, number, distribution and dynamics of these components using transmision electron microscopy (TEM) (Luft 1976; Temmink 1979). Particularly interesting are those methods using particles as markers (e.g. ferritin, gold, viruses, etc) allowing quantitation, at TEM level, of the density and distribution of specific receptor sites. Cytochemical demonstration of cell components requires a balenced fixation of the cells to be studied to preserve both the ultrastructural morphology and the chemical properties of the cell components. The effect of different fixation methods on general ultrastructure of several cell types, including human peripheral blood lymphocytes, has been previously reported (Hayat 1970; Carson et al. 1972; Glauert 1974; Renau-Piqueras etal. 1980a, b, c) but very few reports, using quantitative cytochemical methods, have been * To whom offprint requests should be sent

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f o c u s e d o n t h e i n f l u e n c e o f d i f f e r e n t f i x a t i v e s o n t h e l a b e l i n g o f t h e cell c o a t g l y c o c o n j u g a t e s . I n t h i s p a p e r we r e p o r t t h e e f f e c t o f s e v e r a l f i x a t i v e s o l u t i o n s , commonly used on electron microscopy studies on lymphocytes, on the number o f C o n - A r e c e p t o r sites o f h u m a n p e r i p h e r a l b l o o d T - l y m p h o c y t e s u s i n g g o l d as t h e d e n s e m a r k e r ( A c k e r m a n a n d F r e e m a n 1 9 7 9 ; H e r n g n d e z - Y a g o e t al. 1980).

Materials and Methods Lymphocytes, Human peripheral blood lymphocytes were obtained as previously described (RenauPiqueras et al. 1980a, c). Differential counts showed 96% lymphocytes and cell viability, checked by trypan blue exclusion, was over 95%. T-lymphocytes were obtained by filtration of isolated lymphocytes through a nylon wool column (Greaves and Brown 1974; Renau-Piqueras et al. 1981). The effluent cell population contained over 90% E-rosetting cells (Renau-Piqueras and Cervera 1979).

Fixatives. Glutraldehyde (G) and formaldehyde (F) in aqueous solution were obtained from Tousimis Scientific Products (Glutaraldehyde 25%, Ultrapure TEM Grade, Cat. 1057; Formaldehyde 20%, Ultrapure TEM Grade, Cat. 1009). Osmium tetroxide (Os) was from Merk (Osmium (VIII)-oxid, Cat. 24505). Purity of G was determined measuring the relative values of UV absorbance between 210 and 310 nm using a Zeiss M4QIII spectrophotometer (Fahimi and Drochmans 1965; Gillet and Gull 1976). The fixative solutions used in this study are summarized in Table 1 and were prepared immediately before use in S6rensen phosphate buffer (pH 7.4). Osmolarity was measured using a Knauer-Halbmikro osmometer. Buffer osmolarity was calculated using the criterium of Mathieu et al. (1978) which has been shown to be optimal for the estimation of this parameter in morphological studies on human peripheral blood lymphocytes (Renau-Piqueras et al. 1980a). For preparing mixed fixatives containing Os, solutions of G, F and Os were prepared separately and kept in an ice bath for 30 rain; the mixtures were made at 0~ immediately before use. In Table 1, the final concentration of each fixing agent in the mixture is expressed as percent.

Table 1. Composition, concentration and osmolarities of the different fizative solutions used ~ Fixative

1) 2) 3) 4) 5) 6) 7)

i% G 1% F 0.8% G + 0 . 8 % 1% Os 0.7% Os+0.8% 0.7% Os+0.8% 0.7% Os+0.8%

Mr

(mOsm)

Mb + Sucrose (mOsm)

160 89 89 206 206 89 89

168.7 136.1 115.9 283.0 206.0 130.0 89.0

264.9 331.1 359.9 310.0 310.7 310.0 353.0

Mo

Mf

Buffer

Mb

(mOsm)

(mOsm)

(Molarity)

96.2 195.0 244.0 27.0 104.7 180.0 264.0

0.075 0.04 0.04 0.1 0.1 0.04 0.04

310 310 Fb 310 310 Gc 310 F 310 G + 0 . 8 % F d 310

(mOsm)

Me=effective osmolarity of the fixative solution. Mf=fixative osmolarity (in aqueous solution). Mb = buffer osmolarity. M r = total osmolarity of the fixative solution. Buffer osmolarity was calculated according to Mathieu et al. (I978). Percentages represent the final concentrations of fixing agents. (G, glutaraldehyde; F, formaldehyde; Os, Osmium tetroxide) S6rensen phosphate buffer, pH 7.4; Fixative solutions contain 2 mM MgC12 b Fixative of Karnovsky (1965), modified ~ According to Hirsch and Fedorko (1968), modified a According to Pollard and Ito (1970), modified

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Con-A Gold Labeled Horseradish Peroxidase Labeling. Colloidal gold was prepared by reducing tetrachloroauric acid (Merck) with sodium citrate according to Frens (1973). Preparation of horseradish peroxidase (HRP) colloidal gold complex was according to Ackerman and Freeman (1979). Lymphocytes were fixed in suspension with the fixatives described above for 30 rain at 4~ C. Cells were centrifuged (300 g, 10 rain) and washed, first in 0.1 M S6rensen phosphate buffer (pH 7.4, 310 mOsm, 2 mM MgC12) and then two times in 4% polyvinylpyrrolidone (PVP) in the same buffer. Aliquots containing 108 cells/ml were incubated with shaking at room temperature for 45 rain in Con-A (1.0 mg/ml) (Miles Labs.) in 0.1 M S6rensen phosphate buffer (pH 7.4) containing 4% PVP, incubated in 0.5% HRP-gold reagent for 60 min at room temperature, rinsed three times in 4% PVP in 0.1 M S6rensen phosphate buffer and resuspended in 15% BSA. Then, a BSA-25% G pellet was prepared (Moyne 1974; Renau-Piqueras et al. 1980a) and minced finely. Those ceils fixed with aldehyde solutions were postfixed in 1% Os in 0.28 M veronal acetate buffer (pH 7.4, final osmolarity 310 mOsm) for 45 rain at 4~ C. Cells were postfixed in 0.5% uranyl acetate for 40 min at room temperature, dehydrated in a graded series of acetones (from 50 to 100%) at 0-4 ~ C for 5 min each and embedded in Epon. Controls included the addition of c~-methylD-mannoside (Sigma, final concentration 0.2 M) to the Con-A incubation medium and omission of Con-A from the reaction sequence (Ackerman and Freeman 1979). All samples were processed simultaneously and the same HRP-gold conjugate was used throughout. Ultrathin sections were obtained at the same speed in a given ultratome by the same operator and only silver sections were used for counting purposes. These precautions should minimize errors due to different section thickness and from different proportions of unconjugated colloidal gold. Gold particles were counted on over 40 electronmicrographs (in each case) of whole ceils. Lengths of the plasma membrane countour were measured with a map curvimeter.

Results and Discussion F i x a t i o n affects p r e s e r v a t i o n o f m e m b r a n e c h e m i c a l c o m p o n e n t s ( G l a u e r t 1974; R e n a u - P i q u e r a s et al. 1980b); d i s t r i b u t i o n o f i n t r a m e m b r a n o u s particles (Nerm u t a n d W a r d 1974; Stolinski et al 1977; H a y a n d H a s t y 1979; S c h m a l b r u c h 1980; R e n a u - P i q u e r a s et al. 1980b, c); d i s t r i b u t i o n a n d size o f microvilli ( K n u t t o n et al. 1976; R e n a u - P i q u e r a s and K n e c h t 1979); lectin a g g l u t i n a b i l i t y o f different cells ( I n b a r et al. 1973; V l o d a v s k y et al. 1973) a n d d i s t r i b u t i o n o f m e m b r a n e r e c e p t o r s for different substances (De Petris 1978). Therefore, it a p p e a r e d w o r t h w h i l e to a n a l y z e h o w different fixative solutions c o u l d affect the labeling o f cell c o a t c o m p o n e n t s . In this study we have investigated the effect o f different fixative solutions on the labeling density o f C o n - A r e c e p t o r sites as p a r t o f a s t u d y direct t o w a r d establishing the o p t i m a l p r e p a r a t o r y c o n d i t i o n s for p r e s e r v i n g h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s for electron m i c r o s c o p y studies ( R e n a u - P i q u e r a s a n d K n e c h t 1979; R e n a u - P i q u e r a s et al. 1980 a, b, c). T - l y m p h o c y t e s fixed as d e s c r i b e d a b o v e showed gold particles on their surfaces. All l y m p h o c y t e s in a given section were labeled and, in c o n t r a s t , c o n t r o l s s h o w e d no surface labeling (Fig. 1). E l e c t r o n dense gold particles were easily visible at low m a g n i f i c a t i o n s (Figs. 2, 3). G o l d particles served as m a r k e r s o f C o n - A reactive sites o n the cell surface since no g o l d labeling was o b s e r v e d in o t h e r cell c o m p a r t m e n t s a n d there was no b a c k g r o u n d due to a t t a c h m e n t o f gold to the B S A e m b e d d i n g m e d i u m . The d i s t r i b u t i o n o f gold particles on the T - l y m p h o c y t i c surface a p p e a r e d quite r a n d o m . H o w e v e r , in u r o p o d f o r m i n g cells, gold particles o c c u r e d p o l a r i z e d a l o n g the u r o p o d o p p o s i t e p l a s m a m e m b r a n e (Fig. 3). This c o n f i r m s previous o b s e r v a t i o n s m a d e by others for C o n - A a n d o t h e r r e c e p t o r sites in m o u s e t h y m o c y t e s using ferritin as m a r k e r (De Petris 1978).

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Concanavalin-A Labeling and Fixatives Table 2. Number of gold particles on human T-lymphocytic membranes after different fixative treatment a

* Significant differences, using the Student-t test, were found in all cases except when comparing: 1) F and (Os+G); 2) F and (Os+G+F), and 3) (Os + G) and (Os + G + F). (Number of cells counted for each treatment = 40 ; P < 0.01)

563

Fixative

Mean particle/~tm

+ SD

1) 2) 3) 4) 5) 6) 7)

1.7 4.0 2.4 10.0 4.0 14.6 4.7

1A 1.3 1.2 2.9 2.i 3.7 2.4

G F G+F Os Os+G Os+F Os+G+F

The extent o f surface labeling o f T-lymphocytes after the different fixation procedures was quantified by counting the n u m b e r of gold particles in direct apposition to cell membranes and expressed as the n u m b e r of gold particles per g m lymphocytic surface. Occasionaly, we have f o u n d gold particles near although not touching a cell m e m b r a n e (Fig. 2). These particles were also c o u n t e d because the controls showed no labeling. C o m p a r i s o n o f particle counts f r o m different samples fixed with the same fixative solution under identical conditions showed no significant differences, indicating the reproducibility of the procedure. However, Table 2 shows that with the different fixatives used there are significant differences in the density o f labeling o f the lymphocytic membrane. It is interesting to note that after G fixation, the n u m b e r o f gold particles was lower than after fixation with F or Os. Moreover, G, useed in c o m b i n a t i o n with F or Os, reduced the labeling obtained when the two latter fixatives were used alone. This is particularly interesting since G is the prefered fixative for this kind o f study because o f the excellent ultrastructural preservation obtained (Glauert 1974; Renau-Piqueras et al. 1980a, b, c). This effect can be explained by the fact that G forms crosslinkages in peptide chains (introducing both inter- and intramolecular crosslinks) more realily than F, and F m o r e readily than Os ( H a y a t 1970). Our results (Table 2) are in g o o d agreement with this, showing a close correlation between masking o f receptors sites to the C o n - A complex and cross-linking abilities o f fixatives. Mixtures of aldehydes and Os have been r e c o m m e n d e d for different types o f tissues, particularly for isolated cells and monolayers o f cells ( T r u m p and Bulger 1966; Hirsch and F e d o r k o 1968; Pollard and Ito 1970), The main objection to this type o f fixative ( H o p w o o d 1970; White et al. 1976, 1979; De Bruijn and Den Breejen 1975;

Fig. 1. Human peripheral blood T-lymphocyte. Control (0.2 M c~-methyl-D-mannoside). Cell fixed, before labeling, with 1% glutaraldehyde. Uranyl acetate (before dehydration) and lead citrate staining. No surface labeling, x 26,179 Fig. 2. Human peripheral blood T-lymphocyte. Cell fixed, before labeling, with 0.8% glutaraldehyde plus 0.8% formaldehyde (final concentrations). Uranyl acetate (before dehydration) and lead citrate staining. Gold particles appear only on the cell surface. Occasionaly, we have found gold particles no closely related to cell surface (arrow). x 27,360 Fig. 3. Uropod-forming T-lymphocyte. Fixation as in Fig. 2. Gold particles appear to be preferentially concentrated on the uropod opposite membrane. Arrows show staining artefacts, x 14,140

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D e B r u i j n a n d v a n B u i t e n e n 1980) has b e e n t h a t the fixing a g e n t s p r e s e n t in t h e m i x t u r e r e a c t w i t h e a c h o t h e r a n d , thus, t h e i r p r o p e r t i e s a n d efficiencies m a y be altered. T h i s c o u l d e x p l a i n the o b t a i n e d r e d u c t i o n in s u r f a c e labeling, p a r t i c u l a r l y w h e n u s i n g G in a m i x e d f i x a t i v e c o n t a i n i n g Os ( T a b l e 2). A t any rate, it is c l e a r t h a t n o t all f i x a t i v e s are e q u a l l y a m e n a b l e to q u a n t i t a t i v e studies o n C o n - A r e a c t i v e sites. T h e r e f o r e , the m e t h o d o f f i x a t i o n f o r c y t o c h e m i c a l studies, u s i n g m e t h o d s s i m i l a r to t h o s e d e s c r i b e d here, m u s t be c a r e f u l l y selected a c c o r d i n g to the k i n d o d s t u d y to be p e r f o r m e d . In this, results we r e p o r t here, t o g e t h e r w i t h p r e v i o u s results ( R e n a u - P i q u e r a s a n d K n e c h t 1979; R e n a u P i q u e r a s et al. 1980a, b, c), c a n be useful in selecting the f i x a t i o n p r o c e d u r e f o r c y t o c h e m i c a l a n d u l t r a s t r u c t u r a l studies o n h u m a n p e r i p h e r a l b l o o d l y m p h o cytes.

References Ackerman GA, Freeman WH (1979) Membrane differentiation of developing hemic cells of the bone marrow demonstrated by changes in concanavalin A surface labeling. J Histochem Cytochem 27:1413-1423 Bona C (1975) Physiological significance of the lymphocyte cell coat. Biomedicine 22:97 104 Carson F, Lynn JA, Martin JH (1972) Ultrastructural effect of various buffers, osmolality, and temperature on paraformaldehyde fixation of the formed elements of blood and bone marrow. Tex Rep Biol Med 30:125-142 De Bruijn WC, Den Breejen P (1975) Glycogen, its chemistry and morphological appearence in the electron microscope. II. The complex formed in the selective contrast staining of glycogen. Histochem J 7:205-225 De Bruijn WC, van Buitenen JMH (1980) X-ray microanalysis of aldehyde-fixed glycogen contraststained by OsV[ Fe n and Os w. Ru ~v complexes. J Histochem Cytochem 28:1242-1250 De Petris (1978) Nonuniform distribution of Concanavalin-A receptors and surface antigens on uropod-forming thymocytes. J Cell Biol 79:235 251 Fahimi HD, Drochmans P (1965) Essais de standardisation de la fixation au glutarald6hyde. I. Purification et d6termination de la concentration du glutarald6hyde. J Microscopie 4:725 735 Frens G (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold solutions. Nature (Phys Sci) 241:20-25 Gillet R, Gull K (1976) Glutaraldeyde- Its purity and stability. Histochemie 30:162-167 Glauert AM (1974) Fixation, dehydration and embedding of biological specimens. In: Glauert AM (ed) Pratical methods in electron microscopy, vol 3, Elsevier, Amsterdam Oxford New York, pp 5 72 Greaves MF, Brown G (1974) Purification of human T and B lymphocytes. J Immunol 112:420-423 Hay DH, Hasty DL (1979) Extrusion of particle-free membrane blisters during glutaraldehyde fixation. In: Rash JE, Hudson CS (eds) Freeze-fracture. Methods, artifacts and interpretation. Raven Press, New York, pp 59-66 Hayat MA (1970) Principles and techniques of electron microscopy, vol 1. Van Nostrand Reinhold Company, New York, pp 5-107 Hernb.ndez-Yago J, Knecht E, Grisolia S (1980) (Abstract) Immunocytochemical localization of mitochondrial enzymes. Eur J Cell Biol 22:282 Hirsch JG, Fedorko ME (1968) Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "postfixation" in uranyl acetate. J Cell Biol 38:615-627 Hopwood D (1970) The reactions between formaldehyde, glutaraldehyde and osmium tetroxide, and their fixation effects on bovine serum albumin and on tissue blocks. Histochemie 24: 150-157 Inbar M, Huet C, Oseroff AR, Ben-Bassat H, Sachs L (1973) Inhibition of lectin agglutJnability by fixation of the cell surface membrane. Biochim Biophys Acta 31:594-599 Knutton S, Jackson D, Graham JM, Micklem KJ, Pasternak CA (1976) Microvilli and cell swelling. Nature 262 : 52-54

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Luft JH (1976) The structure and properties of the cell surface coat. Int Rev Cytol 45:291-382 Mathieu O, Claassen H, Weibel ER (1978) Differential effect of glutaraldehyde and buffer osmolarity on cell dimension: A study on lung tissue. J Ultrastruct Res 63:20 34 Moyne G (1974) Mise en evidence de I'ADN par la r6action au Schiff-Thallium. J Microscopie 21:205 208 Nermut MV, Ward BJ (1974) Effect of fixatives on fracture plane in red blood cells. J Microsc (Oxford) 102:29-39 Pollard TD, Ito S (1970) Cytoplasmics filaments of Amoeba proteus. I. The role of filaments in consistency changes and movement. J Cell Biol 46:267-289 Renau-Piqueras J, Cervera J (1979) Alteration of the cell surface morphology in human T-rosetting lymphocytes. An ultrastructural study. Acta Haematol 62:185-190 Renau-Piqueras J, Knecht E (1979) Freeze-fracture and scanning electron microscopy of lymphocytes. Effect of different preparatory techniques on the cell surface morphology. Israel J Med Sci 15:660-667 Reuau-Piqueras J, Miguel A, Knecht E (1980a) Effects of preparatory techniques on the fine structure of human peripheral blood lymphocytes. II. Effect of glutaraldehyde osmolarity. Mikroskopie 36:65 80 Renau-Piqueras J, Knecht E, Miguel A, Hernfindez J (1980 b) (Abstract) Effects of different fixatives on the fine structure of human peripheral blood lymphocytes. In: Broderoo P, de Priester W (eds) Procd. seventh european congress on electron microscopy. The Hague. vol 2. Seventh Eur Congr Electr Microsc Foundt, Leiden, pp 750-75I Renau-Piqueras J, Knecht E, Miguel A, Hernfindez J (1980c) Effects of preparatory techniques on the fine structure of human peripheral blood lympocytes. III. Effect of different fixative solutions. Mikroskopie 38 (In press) Renau-Piqueras J, Martinez-Ramdn A~ Grisolia S (1981) Effects of carbamylation by cyanate on normal and stimulated human peripheral blood lymphocytes. Afr J Clin Exp Immunol 2:137-144 Santer VB (1977) The surface coat of lymphocytes. In: Marchalonis JJ (ed) The lymphocyte. Structure and function. Part II. Marcel Dekker, New York Basel, pp 541-564 Schmalbruch H (1980) Delayed fixation alter the pattern of intramembranous particles in mammalian muscle fibers. J Ultrastruct Res 70:15-20 Stolinski C, Breathnach AS, Bellairs R (1977) Effect of fixation on cell membrane of early embryonic material as observed on freeze-fracture replicas. J Microscopie 112:293 299 Temmink HM (1979) Application of cytochemical methods to electron microscope investigation of cell surface receptors. Biol Cell 36:227 236 Trump BE, Bulger RE (1966) New ultrastructural characterictics of ceils fixed in a glutaraldehyde osmium mixture. Lab Invest 15:368-374 Vlodavsky I, Inbar M, Sachs L (1973) Membrane changes and adenosine triphosphatase content in normal and malignant transformed cells. Proc Natl Acad Sci USA 70:1780 1784 White DL, Andrews SB, Faller JW, Barrnett RJ (1976) The chemical nature of osmium tetroxide fixation and staining of membranes by x-ray photoelectron spectroscopy. Biochim Biophys Acta 436:577 586 White DL, Mazurkiewicz JE, Barrnett RJ (1979) A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures. J Histochem Cytochem 27:1084 1091

Received January 16, 1981 Accepted February 23, 1981