Histochemistry 56, 265-273 (1978) Histochemistry �9 by Springer-Verlag i978

Conjugation of Lectins with Fluorochromes: An Approach to Histochemical Double Labeling of Carbohydrate Components

Jiirgen Rothl, Maximillian Binder 2, and Uwe Jens Gerhard i

1 Institute of Pathology, Friedrich-Schiller University, Ziegelmiihlenweg 1, DDR-69 Jena, German Democratic Republic and 2 Institute for Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria

Summary. Methodical investigations on the coupling of lectins (Con A, LcL, WGA, RcA) to tetramethylrhodamine isothiocyanate (TRITC) are reported. 20~g of TRITC per mg of lectin were found to be the optimal amount of TRITC for the conjugation. With this fluorochrome: protein ratio conjugates were produced which resulted in a specific and brilliant fluorescence in tissue staining. The optimally conjugated lectins were sepa- rated on DEAE-Sephadex-A 50. Using two different lectins which were con- jugated with TRITC or FITC, respectively, a double labeling of different lectin-binding sites in tissue sections was achieved.

Introduction

For the histochemical demonstration of complex carbohydrates various staining reactions such as the alcian blue reaction (Steedman, 1950; Mowry, 1956), the colloidal iron-binding reaction (Hale, 1946; Mtiller, 1955; Graumann and Claus, 1959; Mowry, 1961), the periodic acid-Schiff reaction (McManus, 1946) and others have been developed. A more differentiated visualization of carbohy- drates has been achieved by combination of the above staining reactions (Ritter and Oleson, 1950; Mowry and Winkler, 1956; Spicer, 1960; Spicer and Meyer, 1960; Yamada, 1969, 1970; a.o.). Because of their carbohydrate specificity the lectins have been introduced for histochemical demonstration of sugar moieties (for review see Roth, 1978). There exist various methods applicable for light microscopic visualization of lectin-binding sites: (i) direct staining by means of fluorescent or enzyme-labeled lectins (Smith and Hollers, 1970; Mallucci, 1971; Roth and Thoss, 1974, 1975a, b; Stoddart and Kiernan, 1973; Thoss and Roth, 1975a, b; Etzler and Branstrator, 1974; a.o.), (ii) indirect staining techniques with fluorescent-labeled anti-lectin sera (Comoglio and Gugliehnone, 1972; Mallucci, 1971; Pratt and Gibbson, 1973), and (iii) affinity techniques with fluorescent-labeled markers or horseradish peroxidase (Thoss and Roth, 1976; Kiernan, 1975).

0301-5564/78/0056/0265/$01.80

266 J. Roth et al.:

In t he p r e s e n t s t u d y we r e p o r t o n m e t h p d i c a l i n v e s t i g a t i o n s c o n c e r n i n g

t he c o n j u g a t i o n o f l ec t in s w i t h t e t r a m e t h y l r h o d a m i n e i s o t h i o c y a n a t e ( T R I T C ) .

F u r t h e r m o r e , f l u o r e s c e i n i s o t h i o c y a n a t e ( F I T C ) a n d T R I T C - c o n j u g a t e d l ec t ins

we re u s e d to e s t a b l i s h l i g h t m i c r o s c o p i c d o u b l e l a b e l i n g t e c h n i q u e s f o r t he

d e m o n s t r a t i o n o f spec i f ic s a c c h a r i d e mo ie t i e s .

Materials and Methods

Chemicals: Concanavalin A (Con A) was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). The Ricinus communis agglutinin (RcA) was affinity-purified from saline extracts of Ricinus communis beans by combination of the methods of Nicolson and Blaustein (1972) and Tomita et al. (1972). The Lens culinaris lectin (LcL) was purified from Lens culinaris seeds by the method of Howard and Sage (1969) and that of Toyoshima et al. (1970). The wheat germ agglutinin (WGA) was prepared from dried wheat germs according to Nagata and Burger (1974). FITC was purchased from Feinchemie H. Kallies KG, GDR, and the TRITC from Nordic Immunology (England).

Conjugation of Lectins with TRITC and FITC: A stock solution of TRITC or FITC (1 mg/ml) in dimethylsulfoxide (DMSO) was prepared. The lectin (Con A, LcL, RcA, WGA) was dissolved in 0.5 M carbonate-bicarbonate buffer (pH 9.0) diluted with 9 parts 0.9% NaC1 solution (v/v) with a lectin concentration of 6 mg/ml), This lecfin solution was 0.2 M in respect to the appropriate hapten (~-methyl-D-mannopyranoside for Con A and LcL, D-galactose for RcA and N-acetyl-D- glucosamine for WGA). To this lectin solution an appropriate volume (see results) of the fluoro- chrome stock solution was added dropwise under constant stirring at room temperature. The reaction was allowed to proceed at room temperature for two hours (protected from light).

Purification of the Conjugates: The crude conjugate (approximately 2,2 ml) was transferred to a colunm (0.9 -15 cm) of Sephadex G-25 equilibrated and eluted with phosphate buffered saline (pH 7.2). The first coloured band to emerge was collected and normally used as staining solution after dialysis against PBS. In all cases both a band of intermediate mobility and a band of free dye were observed. In the case of TRITC-conjugated WGA the first band of conjugate eluted from the Sephadex G-25 column (approximately 4 ml) was further fractionated with DEAE-Se- phadex-A 50 (column: 1.5 -15 cm, equilibration and elution with 0.01 M phosphate buffer, pH 8). Fractions of 1.5 ml were collected. The ODsso:OD2s 0 ratio of each fraction was measured with a Unicam SP 800 (Unicam Instruments Ltd., Cambridge, England).

Tissue Preparation and Tissue Staining: Unfixed frozen sections were made from rat myocardium and colon. The sections were mounted on glass slides and covered with the respective lectin conjugate for 30 rain at room temperatme. After two rinses with PBS (5 rain each) the stained sections were covered with PBS-buffered glycerol and examined under the fluorescence microscope Fluoval (VEB Carl Zeiss Jena, GDR).

Double labeling was carried out by subsequent staining with the respective lectin conjugates for 30 rain each.

In the cytochemical controls the lectin conjugates were incubated with the appriate hapten (final 0.2 M) prior to incubation of the tissue sections.

Results

Determination o f the Optimal Amount o f Fluorochrome for Conjugation

C o n A was r e a c t e d w i t h d i f f e r e n t a m o u n t s o f T R I T C (5 gg T R I T C / 1 m g C o n

A, 10 gg T R I T C / 1 m g C o n , A , 15 pg T R I T C / 1 m g C o n A, a n d 20 gg T R I T C /

Lectin Double Labeling Techniques 267

o E o

E E o e4

o "13

o

tn 0

0.6

0.5

o.4

0.3

continuous curve : OD280

interrupted curve :OD550 : OD280

-0.6 OD55 ~

O D280

-0.5

-0.4

-0.3

0.2 J ~ "" ~ ~ -~/~ -0.2

0.1 / 16_ 2 0 ~ 2 I -0.1 4, ,8

ml effluent

Fig. 1. DEAE-Sephadex fractionation of WGA conjugated with suboptimal amounts of TRITC (5 gg TRITC/1 mg WGA). Fraction volume 1,5 ml

0.6

c o n t i n u o u s curve : OD280

o i n te r rup ted curve : O D 5 5 0 : 0 D 2 8 0 o 0 . 5 . 0 . 5

E / o d /

E- 0.4 -0.4 o

oJ

0 . 3 -o.3

09

-o 0.2 -0.2 o

tn

o 0 . I , 0 . 1

4, 8, j ~,2 l j 6 2~0 2,4 2~8 ml e f f l u e n t

Fig. 2. DEAE-Sephadex fractionation of WGA conjugated with optimal amounts of TRITC (20/.tg TRITC/I mg WGA). Fraction volume 1.5 ml

- 0 . 6 0 D55.____2 ~

OD280

268 J. Roth et al. :

1 mg Con A). Fractionation of the conjugates on Sephadex G-25 gave three coloured bands. The first and second band were used for histochemical purposes. The third band represented the free fluoreochrome. Staining of rat myocardium with the first band (5 lag TRITC/1 mg Con A) resulted in an only weak tissue fluorescence. Bright specific fluorescence was obtained when the conjugate pre- pared by reacting of 20 lag TRITC/1 mg Con A was used. Best results were obtained with a final conjugate concentration of 2 mg TRITC'Con A/ml. A weak but specific fluorescence could be observed when the second band eluted from Sephadex G-25 was used for staining. No fluorescence could be detected when the hapten was added to the conjugate prior to tissue staining.

Based upon the results obtained from the conjugation of Con A with TRITC, other lectin conjugates (LcL, RcA, WGA) were prepared accordingly (20 lag TRITC per 1 mg lectin).

Further fractionation on DEAE-Sephadex-A 50 was done sort out over- or undercoupled conjugate fractions by measuring the OD55o:OD2a0 ratio of each fraction. As can be seen from Figure 1, an initial coupling ratio of 5 lag of TRITC per 1 mg WGA gave suboptimal conjugates, whereas 20 lag of TRITC per 1 mg WGA gave optimal conjugates with an ODsso:ODzso ratio of 0.5 (Fig. 2). Conjugation of lectins with FITC was done as described elsewhere (Roth and Thoss, 1975a, b) or by reacting of 20 lag of FITC dissolved in DMSO with 1 mg of lectin.

Tissue Staining

Staining with conjugates of TRITC and FITC with the same lectin gave identical results (Fig. 3). So both TRITC- and FITC-conjugated Con A led to a bright yellow-green or red-orange fluorescence of blood vessels, perivascular connective tissue and of the surface of muscle fibres. Additionally a weak sarcoplasmic fluorescence was observed. Sections from the colon stained with TRITC- or FITC-conjugated WGA exhibited a brilliant fluorescence of goblet cells whereas connective tissue, basement membranes, intestinal epithelia and smooth muscle fibres were only weakly fluoresceing (Fig. 3a, b). Regarding the other lectins, TRITC and FITC conjugates of the same lectin always led to identical fluores- cence patterns (Fig. 3 c, d). In all cases mentioned the addition of the respective hapten inhibited the staining.

Positive results were obtained from the double labeling experiments (Fig. 4). Staining of sections from rat myocardium with TRITC-Con A in the first step and with FITC-WGA in the second step resulted in a spotty red-orange/yellow-green fluorescence. Even more impressive was double labeling of colon sections. Combination of TRITC-WGA and FITC-Con A showed red-orange fluores- cence of goblet cells and extracellular mucus, whereas connective tissue and muscle fibres fluoresced predominantly yellow-green.

A well defined double labeling of tissue sections was achieved also with other staining combinations (WGA-RcA, WGA-Con A, LcL-WGA, LcL-RcA). Addition of the appropriate haptens to the conjugates always strongly suppressed fluorescence.

Fig. 3A-D. Rat colon. Staining with TRITC-conjugated W G A (A) or FITC-conjugated W G A (B) results in a strong fluorescence of goblet cells, whereas the connective tissue, muscle fibres and intestinal epithelia exhibit only a weak fluorescence. Note the identity of the tissue fluorescence pattern. Relatively diffuse fluorescence of all tissue structures of rat coloi1 with TRITC-conjugated Con A (C) or FITC-conjugated Con A (D). Goblet cells are negative. Magnification • 180 (A, C), x 300 (B, D)

270 J. Roth et al. :

Fig. 4. Double staining of rat colon with TRITC-conjugated WGA (orange fluorescence-arrow heads) followed by FITC-conjugated Con A (yellow-green fluorescence-asterisks). Magnification x 490

Discussion

The present results unequivoca l ly demons t r a t e tha t T R I T C can be successfully coup led to va r ious lectins. To our knowledge it was only I n b a r et al. (1973) who h i ther to used T R I T C - c o n j u g a t e d Con A for the label ing of l y m p h o i d

Lectin Double Labeling Techniques 271

cells. In our investigations the compar i son of tissue fluorescence patterns after staining with F I T C - or TRITC-con juga t ed lectins produced identical results. 20 jag/TRITC/1 mg lectin can be considered the optimal amoun t of T R I T C for the conjugat ion with lectins. In contras t to Goding (1976) who reported on a uni form " g r o u n d glass" non-specific staining in controls o f TRITC-con ju - gated IgG we could not observe this p h e n o m e n o n for TRITC-con juga t ed lectins. As recently reported by Goding (1976), D M S O is the best solvent for T R I T C because cont rary to F ITC, T R I T C hardly dissolves in water. The quantities o f D M S O used in our study obviously did not result in an impaired sugar-binding capacity o f lectins. In accordance with Goding (1976) we can confi rm the occur- rence of a band of intermediate mobili ty when separating the conjugates f rom the u n b o u n d f luorochrome by means of c h roma tog raphy with Sephadex G-25. This band has not yet been identified.

By coupl ing o f antibodies and - as demonst ra ted now - of lectins with different f luorochromes, different antigens or lectin-binding sites can be vi- sualized simultaneously in tissue sections or isolated cells. Such double labeling techniques have been applied hitherto for the demonst ra t ion o f different antigens (Frye and Edidin, 1970; Chiappino and Pernis, 1964; Cebra and Goldstein, 1965; Curtain, 1964) or for the detection o f virions (Gour lay and Pemberton, 1971). For the demonst ra t ion o f different lectin-binding sites on the surfacer of one and the same cell electron microscopic double labeling techniques have been developed recently (Roth and Wagner , 1977; Ro th and Binder, 1977). One has to keep in mind that lectins used for double labeling should not react with each other (for example Con A interacts with Ricinus communis aggluti- n i n - P o d d e r et al., 1974; Surolia et al., 1974). The double labeling technique presented in this paper renders possible the demonst ra t ion o f light microscopi- cally different lectin-binding sites (i.e. different sugar residuas) in tissues and cells. In part icular it offers the possibility to study redistribution phenomena of different lectin-binding sites in one and the same cell light microscopically. Fur thermore , the phenomena of reappearance of cell surface carbohydra tes after internalization can be studied.

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Received January 23, 1978