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Thus, avidin-ferritin and streptavidin-gold conjugates have been stable, versatile, and reliable reagents for detection of numerous antibodies to retinal antigens and antigens of other tissues. Their use in diagnostic pathology is expanding new areas of investigation.
[42] V i s u a l i z a t i o n o f I n t r a c e l l u l a r T r a f f i c k i n g o f P r o t e i n s
By
R A N D A L E . MORRIS a n d CATHARINE B . SAELINGER
The intracellular trafficking of proteins following internalization by eukaryotic cells is a subject of intense scientific interest. Unfortunately, few proteins have sufficient electron density to be visualized at the ultrastructural level. To visualize intracellular trafficking, therefore, the protein of interest must be directly conjugated to an electron-dense marker. Alternatively, the protein can be localized by various immunological methods in which the antibody molecules are labeled with an electrondense marker. Because of their superb electron density, gold colloids are the preferred marker. However, direct conjugation of certain proteins to gold colloids results in an aberrant intracellular routing. ~-3 In order to circumvent this problem, we and others have found that the use of goldlabeled antibody techniques is most effective when used in conjunction with postembedding methods. 4 The disadvantage of the postembedding method is that, in most instances, fixation with osmium tetroxide must be omitted. This results in reduced specimen contrast, which makes interpretation difficult. In this chapter we describe a method in which the intracellular routing of protein ligands can be followed by conventional electron microscopy. The technique described permits the intracellular visualization of biotinylated proteins when used in conjunction with avidin-gold colloids. We have successfully used this technique to follow the intracellular routing of
M, C. Willingham, J. A. Hanover, R. B. Dickson, and I. Pastan, Proc. Natl. Acad. Sci. U.S.A. 81, 75 (1984). 2 M. R. Neutra, A. Ciechanover, L. S. Owen, and H. F. Lodish, J. Histochem. Cytochem. 33, 1134 (1985). 3 B. van Deurs, T. I. Tonnessen, O. W. Petersen, K. Sandvig, and S. Olsnes, J. Cell Biol. 102, 37 (1986). 4 M. C. Willingham, J. Histochem. Cytochem. 31, 791 (1983).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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several proteins. We suggest that this technique can be tailored to a variety of proteins and cell types. Biotinylation o f Proteins
The biotinylation of proteins is a mild procedure involving the nucleophilic attack on proteins by the N-hydroxysuccinimide ester group of biotinyl-N-hydroxysuccinimide ester (BNHS; Pierce Chemical Co., Rockford, IL). The procedure described below results in very stable products that retain good biological activity. 1. Dialyze the protein overnight at 4° against 50 mM borate buffer (pH 9.1; 19.06 g/liter Na2B407 • 10H20). We usually use 1 mg of protein in a volume of 1 ml and dialyze against 1 liter of buffer. 2. The biotinylation is done at a 5 : 1 molar ratio (BNHS/protein) and a I : 50 volume ratio (BNHS/protein) as described by Bayer et al. 5 Calculate the required amount of BNHS and dissolve in dimethylformamide (DMF). Since only 20 ~1 will be required for a 1 ml protein solution, we typically prepare 0.5 ml of DMF containing BNHS. 3. Place the dialyzed protein solution in a glass test tube and clarify by centrifugation if necessary. To the solution add the appropriate volume of DMF containing BNHS. 4. Incubate at 23 ° for 4 hr. 5. Dialyze the resultant solution against phosphate-buffered saline (pH 7.4, PBS; 8.01 g/liter NaCI, 1.18 g/liter Na2HPO4, 0.22 g/liter KC1, 0.26 g/liter K2HPO4- H20). 6. Store biotinyl proteins at 4 ° without azide or other anti-microbials. For long-term storage, they can be stored at - 2 0 ° without deleterious effects. This procedure results in biotinylation preferentially via e-amino groups of lysyl residues. However, this can result in loss of biological activity if the lysine groups are required for specific activity. In such an event, the procedure can be modified to direct the nucleophilic attack to the a-amino groups. In this instance, the reaction is done between pH 5.0 a n d 6.0. 5,6
Using the procedure described we have found that an average of 2.5 mol of biotin is incorporated for every mole of protein, i.e., Pseudomonas 5 E. A. Bayer, E. Skutelsky, and M. Wilchek, this series, Vol. 62, p. 308. 6 p. Cuatrecasas and I. Parikh, Biochemistry 11, 2291 (1972).
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exotoxin A. 7 (This is estimated by the method of Means and Feany 8 using 2,4,6-trinitrobenzenesulfonic acid.) Higher molar ratios (BNHS/protein) can be used to increase the level of incorporation. It has been our experience, however, that the higher the molar ratio, the greater the reduction in biological activity. We therefore rarely exceed a 5 : 1 (initial) molar ratio.
Preparation of Avidin-Gold Colloids The interaction between egg-white avidin and biotin is among the most avid interactions known (Kd 10-15 M ) . 9 The binding of avidin to biotin is several orders of magnitude greater than that found for most antigenantibody interactions and can be considered essentially an irreversible noncovalent interaction. This has important ramifications in the use of biotinyl protein-avidin-gold to study intracellular trafficking. Following internalization, ligands are routed through various acidic compartments, e.g., endosomes and lysosomes (pH 4.5-5.5), where certain receptors and ligands are known to dissociate. 1° The interaction between avidin and biotin is stable at these pH values,9 and therefore one is assured of following the labeled ligand and not dissociated gold colloids. Gold colloids of varying sizes can be easily prepared by the reduction of gold chloride. Colloids with average diameters ranging from 5 to 20 nm are routinely used for electron microscopy. It has been our experience that only 5- to 7-nm avidin-gold colloids can be used in the biotinyl ligand-avidin-gold method; larger size colloids, i.e., 12 and 18 nm, do not bind biotinyl ligands. The reasons for this are not known but possibly result from steric hindrance. Described below is a method used for the production of 5-nm gold sols by reduction of gold chloride with white phosphorus, i1 1. To a 500-ml round-bottomed flask add 240 ml of doubly distilled water, 5.4 ml of 0.1 M K2CO3 and 6 ml of 0.5% gold chloride solution (Fisher Scientific, Fair Lawn, N J). 2. Prepare a solution of white phosphorus-saturated diethyl ether. Saturation is complete when the solution has a distinctively gray color. Note: Unused white phosphorus-saturated ether is neutralized by the addition of an equal volume of 1% CuSO4. 7 R. E. Morris and C. B. Saelinger, J. Histochern. Cytochem. 32, 124 (1984). s G. E. Means and R. E. Feany, in "Chemical Modification of Proteins." Holden-Day, San Francisco, California, 1971. 9 N. M. Green, Adv. Protein Chem. 29, 85 (1975). 10 H. J. Geuze, J. W. Slot, G. J. A. M. Strous, H. F. Lodish, and A. L. Schwartz, Cell 32, 277 (1983). i1 M. Horisberger, Biol. Cell. 36, 253 (1979).
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3. Initiate the reduction of the gold chloride solution by adding of 2 ml of a 1:5 white phosphorus-ether solution (1.6 ml of diethyl ether and 0.4 ml of white phosphorus-saturated ether). 4. Incubate the solution at 23° for 30 min, during which time it becomes a rust-brown color. 5. The reaction is driven to completion by refluxing. The end point of the reaction is noted when the solution becomes deep red in color. This usually requires 5-20 min of refluxing. The gold sols are cooled to 23 °, pH adjusted (see below), and stabilized with avidin, i.e., converted to gold colloids. Five-nanometer gold sols can also be produced by reduction of gold chloride with sodium citrate and tannic acid. 12,13We advocate preparation with white phosphorus because we have experienced some reduction in the recognition of biotinyl ligand when using avidin-gold colloids prepared by the sodium citrate-tannic acid method. This may result because tannic acid is a mordant, and residual acid may denature the avidin.14 After the gold sols are prepared they are stabilized, i.e., converted to gold colloids, by the adsorption of avidin onto the sols. Stabilization occurs as the result of electrostatic interactions between the gold sols and the protein being adsorbed. Handley et al. ~5have shown that this interaction is very stable under a variety of conditions. We have used two sources of avidin with equal success, egg-white avidin and streptavidin. Egg-white avidin is a basic protein with a pI of 10.5.9 At physiological pH, the protein carries a net positive charge and nonspecifically binds to cell membranes. For this reason egg-white avidin should be succinylated prior to use (see below). Streptavidin, on the other hand, has a neutral pI and requires no modification prior to use. Streptavidin is a protein secreted from S t r e p t o m y c e s avidinii that has essentially the same molecular weight, subunit structure, and binding affinity as the egg-white protein.16 Because gold sols are susceptible to flocculation by the presence of ions, all proteins to be labeled with gold must be exhaustively dialyzed against low-molarity buffer prior to adsorption. Furthermore, the greatest stability of the colloids occurs when adsorption is carried out at a pH value slightly above the pI of the protein being adsorbed. ~7Accordingly, avidin 12 H. Muhlpfordt, Experientia 38, 1127 (1982). 13 j. Slot and H. J. Geuze, Eur. J. Cell Biol. 38, 87 (1985). 14 K. Aoki, S. Kajiwara, R. Shinke, and H. Nishira, Anal. Biochem. 95, 575 (1979). 15 D. A. Handley, C. M. Arbeeny, L. D. Witte, and S. Chien, Proc. Natl. Acad. Sci. U.S.A. 78, 368 (1981). 16 L. Chalet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964). i7 W. D. Geoghegan and G. A. Ackerman, J. Histochem. Cytochem. 11, 1187 (1977).
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is dialyzed against repeated changes of 5 mM phosphate buffer (pH 7.5; 0.11 g/liter NaHPO4. H20, I. 13 g/liter Na2HPO4" 7H20). Typically we dialyze against 4 changes of buffer for 40 hr at 4 ° prior to adsorption.
Succinylation of Egg-White Avidin 1. Dissolve 10 mg of egg-white avidin (Sigma Chemical Co., St. Louis, MO) in 10 ml o f a 3 M sodium acetate solution. Add 10 mg of succinic anhydride; incubate overnight at 4 °. 2. Incubate for 1 additional hour at 23 °. Place the solution into a dialysis bag; allow sufficient space for the volume to double. 3. Dialyze against 5 mM phosphate buffer (pH 7.5) as described above. The amount of avidin required to stabilize a known volume of gold sols is determined by the method of Geoghegan and Ackerman.~7 1. Adjust the pH of the gold sols to 7.5 using either 0. ! M K2CO3 or 1% acetic acid. (Note: Submersion of the pH electrode into the gold sol solution will result in short-circuiting of the electrode. To avoid this, take a 5-ml sample of the sols and add 10 drops of a 1% polyethylene glycol solution (MW ->20,000) prior to immersion of the electrode. This will stabilize the sols and prevent damaging the electrode.) 2. To 10 plastic test tubes add 1 ml of the 5 mMphosphate buffer (pH 7.5). Leave the first tube as a control tube; starting with the second tube, serially dilute the avidin solution ! : 2, 1 : 4, 1 : 8, etc. 3. Add 5 ml of the pH-adjusted gold sols to each tube, mix well. Allow all tubes to stand for at least 1 min at 23 °. 4. Add 1 ml of a 10% NaCI solution to each tube. This will cause flocculation, detected by a change in color from red to blue, in those samples which have not been stabilized by the protein. We typically allow 30 rain for the reaction to go to completion. 5. To determine the end point of the reaction, that is, the greatest dilution which stabilizes the gold against flocculation, visually identify the tube with the greatest dilution that retains a clear red color. To guard against possible errors in dilution, we use twice the amount of protein indicated by the test. If, for example, the dilution assay indicated that stabilization occurs at 1 : 16, then we would use 1 : 8 as the stabilizing dilution. If we want to prepare 100 ml of avidin-stabilized gold, we would add 2.5 ml of undiluted protein [volume desired/(dilution factor x volume of gold stabilized in the test, i.e., 5)].
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6. The protein is then added (concentration -< 1 mg/ml in 5 mM phosphate buffer) to a rapidly stirring solution of pH-adjusted gold sols. 7. After stirring for 15 min, a 5-ml sample is taken and tested for stability as in step 4. 8. The avidin-gold colloids are washed by centrifugation at 18,000 g for 3 hr at 4 °. This results in the formation of a loose pellet and a tight pellet. The supernatant fluid is discarded, and the loose pellet is resuspended to the original volume in 5 mM phosphate buffer (pH 7.5) and transferred to another centrifuge tube. The remaining tight pellet is discarded. 9. The gold colloids are repelleted by a second centrifugation as above. The resulting loose pellet is resuspended in sufficient 5 mM phosphate buffer (pH 7.5) such that its color approximates that of the initial solution, i.e., step 7 (above). The resultant avidin-gold colloidal suspension is transferred to a 100-ml beaker and stirred. While stirring, an equal volume of double-strength stabilizing buffer [18.0 g/liter NaCI, 12.1 g/liter Tris-HC1, 400 mg/liter polyethylene glycol (MW >20,000) (pH 7.5)] is added. 10. After 15 min of stirring, the avidin-gold colloids are placed in plastic tubes and stored at 4 ° until needed.18 The procedure described varies slightly from previous published methods. 7,19 It is important to note that we use the avidin-gold colloids within 1 month after production. Avidin-gold colloids older than 1 month have an increased tendency to bind nonspecifically to cells. Just before use, the avidin-gold colloids are absorbed against the cell line being studied. For example, 5 ml of avidin-gold colloids is absorbed against nearly confluent monolayers of mouse LM fibroblasts. Prior to absorption the LM cells are exhausted of exogenous biotin and cooled to 4°. Absorption is repeated 3 times for 30 rain at 4°. Large aggregates and debris are removed by centrifugation at 18,000 g for 20 min at 4 °.
Biotinyl Ligand-Avidin-Gold Method The procedure described is limited to in vitro conditions. This restriction is due to the significant concentrations of biotin present in the sera of 18 Since we usually u s e the a v i d i n - g o l d colloids within I m o n t h of production, we do not add azide or similar agents. In a n y case, for internalization studies (as well as for other studies in which viable cells are used), it is, o f course, not r e c o m m e n d e d to u s e a n y s u c h toxic antimicrobial agents. 19 R. E. Morris and C. B. Saelinger, Infect. Immun. 52, 445 (1986).
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animals. 2° This fact also dictates that, during the experimentation procedures, medium containing biotin or animal sera must not be used. Typically, we grow cells in complete medium with I0% sera. For 2 hr prior to the initiation of the experiment we exhaust the cells of exogenous biotin by incubation at 37° in HEPES-buffered Hanks' balanced salt solution (HBSS). Failure to include this step results in high levels of nonspecific binding by the avidin-gold colloids. The steps in the procedure are given below. 1. Wash all samples 3 times with cold HBSS; incubate at 4 ° for 15 min. 2. Add the biotinylated protein to the samples in a minimal volume of HBSS; incubate at 4 ° for 30 min. 21 3. Wash all samples 3 times with cold HBSS. 4. Add the avidin-gold conjugates in a minimal volume of HBSS to each sample; incubate for 30 min at 4 °. 5. To those samples which are to be warmed, add prewarmed HBSS and incubate on a warming plate. After appropriate incubation, wash samples 3 times with cold HBSS. 6. Wash all samples twice with cold 0.1 M sodium cacodylate buffer (pH 7.4) containing 0.05% CaC12 and 5.0% sucrose (SCB). 7. Fix all samples with SCB containing 2.0% paraformaldehyde and 2.5% (w/v) glutaraldehyde; fix for 30 min at 4 °. 8. Repeat step 6. 9. Post fix all samples with SCB containing 1% osmium tetroxide reduced with 1% potassium ferrocyanide22; fix for 1 hr at 4 °. 10. Repeat step 6. 11. Wash all samples twice with distilled water at 23°. 12. Begin dehydration by 3 washes with 70% (v/v) ethanol. 13. Stain e n b l o c for 10 min with 0.5% uranyl acetate in 70% (v/v) ethanol. 14. Continue dehydration by 2 washes with 70% ethanol followed by 3 washes with absolute ethanol. 15. Infiltrate and embed by standard procedures using epoxy resin. 2o R. E. Morris and C. B. Saelinger, Immunol. Today 5, 127 (1984). 21 C o n c e n t r a t i o n s and v o l u m e s of reagents used in these experiments vary from e x p e r i m e n t to experiment. F o r e x a m p l e , in one instance we m a y add l ml of a given biotinyl protein to a 16 × 85 m m Leighton tube; in a n o t h e r instance we m a y add 2.5 ml to a 100 m m petri dish. C o n c e n t r a t i o n s vary depending on the biotinyl ligand and the cell type. For most e x p e r i m e n t s we u s e L M cells grown on 16 x 85 m m glass Leighton tubes, using a l-ml v o l u m e of biotinyl toxin at concentrations ranging from 1 ng/ml to 1 p.g/ml. 22 M. J. K a r n o v s k y , J. Cell Biol. 51, 146a (1971).
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A control sample in which the biotinyl ligand is omitted should be included. This will measure nonspecific (background) binding of the gold colloid to cells. One of the advantages of the biotinyl ligand-avidin-gold system is that specific binding can be demonstrated by a competition assay. This is done by including a sample with a 100- to 200-fold excess of native ligand (not biotinylated) during the primary incubation step with the biotinyl ligand. The procedure described above is done on glass Leighton tubes. 23 Following polymerization at 60°, the resin containing the monolayer is freed from the glass by submersion in an ice bath. In preparation for sectioning, small areas of the plastic are drilled out with an electric cork borer (E. H. Sargent and Company, Birmingham, AL) and mounted on 3/4 × I/4 inch wooden dowels. 24 Alternatively, the cells can be grown on plastic petri dishes. At the conclusion of step 11, the cells are scraped from the plastic surface, placed in 1% low-temperature gelling agarose (Sigma), and pelleted (in a conical centrifuge tube) by low-speed centrifugation at 23 °. Following overnight solidification at 4°, the cell-rich regions of the agarose are diced into pieces of about 1 mm 3 and treated as described above. The samples are ultimately embedded in epoxy resin in BEEM capsules. The samples are viewed after preparation of ultrathin sections. The above procedure results in sufficient specimen contrast so that the samples do not require staining. Comments
We have used the biotinyl ligand-avidin-gold method to follow the intracellular trafficking of several different proteins. The majority of our experience has been with the intracellular trafficking of Pseudomonas exotoxin A (PE) by mouse LM fibroblasts. We have shown that LM cells, a cell line exquisitely sensitive to PE, internalize biotinyl-PE-avidin-gold by receptor-mediated endocytosis, route the complex to the Golgi region in endosomes, and ultimately deliver the complex to the lysosomal compartment. The entire process takes 20-30 min at 37°. 19,25,26We have also followed the internalization and intracellular trafficking of biotinyl diphtheria toxin by Vero cells, a cell line exquisitely sensitive to this toxin.
23 j. S. Sutton, Stain Technol. 40, 151 (1965). 24 R. E. Morris, G. M. Ciraolo, D. A. Cohen, and H. C. Bubel, In Vitro 16, 136 (1980). 2~ R. E. Morris, M. D. Manhart, and C. 13. Saelinger, Infect. Immun. 40, 806 (1983). 26 R. E. Morris, in "Microbiology 1985" (L. Leive, ed.), p. 91. American Society for Microbiology, Washington, D.C., 1985.
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The routing of diphtheria toxin in Vero cells is similar to that of PE in mouse LM cells. 27 Others have shown that ligands bound to gold are routed differently in cells than are native ligands. For example, van Deurs et alp showed that Vero and MCF-7 cells internalize native ricin by receptor-mediated endocytosis and that the ricin is routed by the endosomal system to the Golgi cisternae. In contrast, ricin-gold conjugates, although internalized by receptor-mediated endocytosis, are not observed in the Golgi. They estimated that each ricin-gold conjugate contains 2 to 4 ricin molecules per gold particle z8 and is therefore polyvalent. They further showed that monovalent conjugates, formed between ricin and horseradish peroxidase, are routed intracellularly as is native ricin. The authors concluded that the valency of the ligand conjugate dictates the intracellular trafficking pattern. Others have reported similar observations with transferringold conjugates. 1,2 Two experimental observations lead us to believe that the biotinyl ligand-avidin-gold technique circumvents this problem. First, biotinyl diphtheria toxin-avidin-gold complexes are routed differently in mouse LM cells than are biotinyl Pseudomonas toxin-avidin-gold complexes. Mouse LM cells are resistant to diphtheria toxin. The diphtheria toxingold conjugates are not internalized by receptor-mediated endocytosis and are not routed to the Golgi region. 29 This occurs in spite of the fact that LM cells have receptors for diphtheria toxin 3° and that diphtheria toxin and Pseudomonas toxin inhibit mammalian cell protein synthesis by identical mechanisms. We feel that this observation supports our hypothesis that the mechanism of entry and intracellular trafficking dictates the susceptibility of a cell to toxin. Second, our morphological observations are corroborated by biochemical data using native toxin. 31,32We feel that the biotinyl ligand-avidin-gold method allows for the ligand to be processed normally because the biotinyl ligand is permitted to interact with its receptor prior to addition of gold. Thus, the initial binding of ligand is not influenced by the gold, i.e., the valency is not altered. In conclusion, we advocate the use of the biotinyl ligand-avidin-gold technique as a tool to visualize intracellular trafficking. There are three 27 R. E. Morris, A. S. Gerstein, P. F. Bonventre, and C. B. Saelinger, Infect. Immun. 50, 721 (1985). z8 B. van Deurs, L. R. Pedersen, A. Sundan, S. Olsnes, and K. Sandvig, Exp. Cell Res. 159, 287 (1985). 29 R. E. Morris and C. B. Saelinger, Infect. Immun. 42, 812 (1983). 3o j. R. Didsbury, J. M. Moehring, and T. J. Moehring, Mol. Cell. Biol. 3, 1283 (1983). 3i C. B. Saelinger, R. E. Morris, and G. Foertsch, Eur. J. Clin. Microbiol. 4, 170 (1985). 3~ C. B. Saelinger and R. E. Morris, Antibiot. Chemother. (Basel) 39, 149 (1987).
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advantages of this technique over other methods for following intracellular trafficking at the ultrastructural level. First, because of the affinity of avidin for biotin, dissociation of the ligand from the electron-dense marker is negligible. Second, competition of native ligand and biotinyl ligand for a receptor on the cell surface can be measured. Third, the technique permits the use of fixation and embedding protocols which result in excellent ultrastructural preservation. This allows for more precise definition of intracellular locations.
[43] L o c a l i z a t i o n o f L e c t i n R e c e p t o r s
By RICHARD M. PINO Introduction The location and chemical composition of cell surface components have often been examined by ultrastructural cytochemical methods.~-5 A basic approach is the identification of monosaccharides using lectins. 6 With direct labeling, cells or tissues are treated with lectins that are coupled to ferritin, 7 which has inherent electron density. Lectins can also be coupled to horseradish peroxidase (HRP), 8 which can generate an electron-dense reaction product after incubation in a cytochemical medium. HRP methods are also applicable to light microscopic cytochemistry analysis. The avidin-biotin method 9 is a reliable indirect method that can be used for lectin affinity 1°,1~ and immunocytochemical 4,1°,12 localizations on i D. Danon, L. Goldstein, Y. Marikovsky, and E. Skutelsky, J. UItrastruct. Res. 38, 500 (1972). 2 p. p. H. DeBruyn and S. Michelson, J. Cell Biol. 82, 708 (1979). 3 E. Essner, R. M. Pino, and R. A. Griewski, Curr. Eye Res. 1, 381 (1981). 4 R. M. Pino, Invest. Ophthalmol. Visual Sci. 27, 840 (1986). 5 R. M. Pino, Cell Tissue Res. 250, 257 (1987). 6 N. Sharon and H. Lis, Science 177, 949 (1972). 7 G. L. Nicolson and S. J. Singer, J. CellBiol. 60, 236 (1974). 8 W. Bernhard and S. Avrameas, Exp. Cell Res. 64, 232 (1971). 9 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). l0 E. A. Bayer, M. Wilchek, and E. Skutelsky, FEBS Left. 68, 240 (1976). H S. M. Hsu and L. Raine, J. Histochem. Cytochem. 30~ 157 (1982). 12 S. M. Hsu, L. Raine, and H. Fanger, J. Histochern. Cytochem. 29, 577 (1981).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Thus, avidin-ferritin and streptavidin-gold conjugates have been stable, versatile, and reliable reagents for detection of numerous antibodies to retinal antigens and antigens of other tissues. Their use in diagnostic pathology is expanding new areas of investigation.
[42] V i s u a l i z a t i o n o f I n t r a c e l l u l a r T r a f f i c k i n g o f P r o t e i n s
By
R A N D A L E . MORRIS a n d CATHARINE B . SAELINGER
The intracellular trafficking of proteins following internalization by eukaryotic cells is a subject of intense scientific interest. Unfortunately, few proteins have sufficient electron density to be visualized at the ultrastructural level. To visualize intracellular trafficking, therefore, the protein of interest must be directly conjugated to an electron-dense marker. Alternatively, the protein can be localized by various immunological methods in which the antibody molecules are labeled with an electrondense marker. Because of their superb electron density, gold colloids are the preferred marker. However, direct conjugation of certain proteins to gold colloids results in an aberrant intracellular routing. ~-3 In order to circumvent this problem, we and others have found that the use of goldlabeled antibody techniques is most effective when used in conjunction with postembedding methods. 4 The disadvantage of the postembedding method is that, in most instances, fixation with osmium tetroxide must be omitted. This results in reduced specimen contrast, which makes interpretation difficult. In this chapter we describe a method in which the intracellular routing of protein ligands can be followed by conventional electron microscopy. The technique described permits the intracellular visualization of biotinylated proteins when used in conjunction with avidin-gold colloids. We have successfully used this technique to follow the intracellular routing of
M, C. Willingham, J. A. Hanover, R. B. Dickson, and I. Pastan, Proc. Natl. Acad. Sci. U.S.A. 81, 75 (1984). 2 M. R. Neutra, A. Ciechanover, L. S. Owen, and H. F. Lodish, J. Histochem. Cytochem. 33, 1134 (1985). 3 B. van Deurs, T. I. Tonnessen, O. W. Petersen, K. Sandvig, and S. Olsnes, J. Cell Biol. 102, 37 (1986). 4 M. C. Willingham, J. Histochem. Cytochem. 31, 791 (1983).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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several proteins. We suggest that this technique can be tailored to a variety of proteins and cell types. Biotinylation o f Proteins
The biotinylation of proteins is a mild procedure involving the nucleophilic attack on proteins by the N-hydroxysuccinimide ester group of biotinyl-N-hydroxysuccinimide ester (BNHS; Pierce Chemical Co., Rockford, IL). The procedure described below results in very stable products that retain good biological activity. 1. Dialyze the protein overnight at 4° against 50 mM borate buffer (pH 9.1; 19.06 g/liter Na2B407 • 10H20). We usually use 1 mg of protein in a volume of 1 ml and dialyze against 1 liter of buffer. 2. The biotinylation is done at a 5 : 1 molar ratio (BNHS/protein) and a I : 50 volume ratio (BNHS/protein) as described by Bayer et al. 5 Calculate the required amount of BNHS and dissolve in dimethylformamide (DMF). Since only 20 ~1 will be required for a 1 ml protein solution, we typically prepare 0.5 ml of DMF containing BNHS. 3. Place the dialyzed protein solution in a glass test tube and clarify by centrifugation if necessary. To the solution add the appropriate volume of DMF containing BNHS. 4. Incubate at 23 ° for 4 hr. 5. Dialyze the resultant solution against phosphate-buffered saline (pH 7.4, PBS; 8.01 g/liter NaCI, 1.18 g/liter Na2HPO4, 0.22 g/liter KC1, 0.26 g/liter K2HPO4- H20). 6. Store biotinyl proteins at 4 ° without azide or other anti-microbials. For long-term storage, they can be stored at - 2 0 ° without deleterious effects. This procedure results in biotinylation preferentially via e-amino groups of lysyl residues. However, this can result in loss of biological activity if the lysine groups are required for specific activity. In such an event, the procedure can be modified to direct the nucleophilic attack to the a-amino groups. In this instance, the reaction is done between pH 5.0 a n d 6.0. 5,6
Using the procedure described we have found that an average of 2.5 mol of biotin is incorporated for every mole of protein, i.e., Pseudomonas 5 E. A. Bayer, E. Skutelsky, and M. Wilchek, this series, Vol. 62, p. 308. 6 p. Cuatrecasas and I. Parikh, Biochemistry 11, 2291 (1972).
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exotoxin A. 7 (This is estimated by the method of Means and Feany 8 using 2,4,6-trinitrobenzenesulfonic acid.) Higher molar ratios (BNHS/protein) can be used to increase the level of incorporation. It has been our experience, however, that the higher the molar ratio, the greater the reduction in biological activity. We therefore rarely exceed a 5 : 1 (initial) molar ratio.
Preparation of Avidin-Gold Colloids The interaction between egg-white avidin and biotin is among the most avid interactions known (Kd 10-15 M ) . 9 The binding of avidin to biotin is several orders of magnitude greater than that found for most antigenantibody interactions and can be considered essentially an irreversible noncovalent interaction. This has important ramifications in the use of biotinyl protein-avidin-gold to study intracellular trafficking. Following internalization, ligands are routed through various acidic compartments, e.g., endosomes and lysosomes (pH 4.5-5.5), where certain receptors and ligands are known to dissociate. 1° The interaction between avidin and biotin is stable at these pH values,9 and therefore one is assured of following the labeled ligand and not dissociated gold colloids. Gold colloids of varying sizes can be easily prepared by the reduction of gold chloride. Colloids with average diameters ranging from 5 to 20 nm are routinely used for electron microscopy. It has been our experience that only 5- to 7-nm avidin-gold colloids can be used in the biotinyl ligand-avidin-gold method; larger size colloids, i.e., 12 and 18 nm, do not bind biotinyl ligands. The reasons for this are not known but possibly result from steric hindrance. Described below is a method used for the production of 5-nm gold sols by reduction of gold chloride with white phosphorus, i1 1. To a 500-ml round-bottomed flask add 240 ml of doubly distilled water, 5.4 ml of 0.1 M K2CO3 and 6 ml of 0.5% gold chloride solution (Fisher Scientific, Fair Lawn, N J). 2. Prepare a solution of white phosphorus-saturated diethyl ether. Saturation is complete when the solution has a distinctively gray color. Note: Unused white phosphorus-saturated ether is neutralized by the addition of an equal volume of 1% CuSO4. 7 R. E. Morris and C. B. Saelinger, J. Histochern. Cytochem. 32, 124 (1984). s G. E. Means and R. E. Feany, in "Chemical Modification of Proteins." Holden-Day, San Francisco, California, 1971. 9 N. M. Green, Adv. Protein Chem. 29, 85 (1975). 10 H. J. Geuze, J. W. Slot, G. J. A. M. Strous, H. F. Lodish, and A. L. Schwartz, Cell 32, 277 (1983). i1 M. Horisberger, Biol. Cell. 36, 253 (1979).
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3. Initiate the reduction of the gold chloride solution by adding of 2 ml of a 1:5 white phosphorus-ether solution (1.6 ml of diethyl ether and 0.4 ml of white phosphorus-saturated ether). 4. Incubate the solution at 23° for 30 min, during which time it becomes a rust-brown color. 5. The reaction is driven to completion by refluxing. The end point of the reaction is noted when the solution becomes deep red in color. This usually requires 5-20 min of refluxing. The gold sols are cooled to 23 °, pH adjusted (see below), and stabilized with avidin, i.e., converted to gold colloids. Five-nanometer gold sols can also be produced by reduction of gold chloride with sodium citrate and tannic acid. 12,13We advocate preparation with white phosphorus because we have experienced some reduction in the recognition of biotinyl ligand when using avidin-gold colloids prepared by the sodium citrate-tannic acid method. This may result because tannic acid is a mordant, and residual acid may denature the avidin.14 After the gold sols are prepared they are stabilized, i.e., converted to gold colloids, by the adsorption of avidin onto the sols. Stabilization occurs as the result of electrostatic interactions between the gold sols and the protein being adsorbed. Handley et al. ~5have shown that this interaction is very stable under a variety of conditions. We have used two sources of avidin with equal success, egg-white avidin and streptavidin. Egg-white avidin is a basic protein with a pI of 10.5.9 At physiological pH, the protein carries a net positive charge and nonspecifically binds to cell membranes. For this reason egg-white avidin should be succinylated prior to use (see below). Streptavidin, on the other hand, has a neutral pI and requires no modification prior to use. Streptavidin is a protein secreted from S t r e p t o m y c e s avidinii that has essentially the same molecular weight, subunit structure, and binding affinity as the egg-white protein.16 Because gold sols are susceptible to flocculation by the presence of ions, all proteins to be labeled with gold must be exhaustively dialyzed against low-molarity buffer prior to adsorption. Furthermore, the greatest stability of the colloids occurs when adsorption is carried out at a pH value slightly above the pI of the protein being adsorbed. ~7Accordingly, avidin 12 H. Muhlpfordt, Experientia 38, 1127 (1982). 13 j. Slot and H. J. Geuze, Eur. J. Cell Biol. 38, 87 (1985). 14 K. Aoki, S. Kajiwara, R. Shinke, and H. Nishira, Anal. Biochem. 95, 575 (1979). 15 D. A. Handley, C. M. Arbeeny, L. D. Witte, and S. Chien, Proc. Natl. Acad. Sci. U.S.A. 78, 368 (1981). 16 L. Chalet and F. J. Wolf, Arch. Biochem. Biophys. 106, 1 (1964). i7 W. D. Geoghegan and G. A. Ackerman, J. Histochem. Cytochem. 11, 1187 (1977).
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is dialyzed against repeated changes of 5 mM phosphate buffer (pH 7.5; 0.11 g/liter NaHPO4. H20, I. 13 g/liter Na2HPO4" 7H20). Typically we dialyze against 4 changes of buffer for 40 hr at 4 ° prior to adsorption.
Succinylation of Egg-White Avidin 1. Dissolve 10 mg of egg-white avidin (Sigma Chemical Co., St. Louis, MO) in 10 ml o f a 3 M sodium acetate solution. Add 10 mg of succinic anhydride; incubate overnight at 4 °. 2. Incubate for 1 additional hour at 23 °. Place the solution into a dialysis bag; allow sufficient space for the volume to double. 3. Dialyze against 5 mM phosphate buffer (pH 7.5) as described above. The amount of avidin required to stabilize a known volume of gold sols is determined by the method of Geoghegan and Ackerman.~7 1. Adjust the pH of the gold sols to 7.5 using either 0. ! M K2CO3 or 1% acetic acid. (Note: Submersion of the pH electrode into the gold sol solution will result in short-circuiting of the electrode. To avoid this, take a 5-ml sample of the sols and add 10 drops of a 1% polyethylene glycol solution (MW ->20,000) prior to immersion of the electrode. This will stabilize the sols and prevent damaging the electrode.) 2. To 10 plastic test tubes add 1 ml of the 5 mMphosphate buffer (pH 7.5). Leave the first tube as a control tube; starting with the second tube, serially dilute the avidin solution ! : 2, 1 : 4, 1 : 8, etc. 3. Add 5 ml of the pH-adjusted gold sols to each tube, mix well. Allow all tubes to stand for at least 1 min at 23 °. 4. Add 1 ml of a 10% NaCI solution to each tube. This will cause flocculation, detected by a change in color from red to blue, in those samples which have not been stabilized by the protein. We typically allow 30 rain for the reaction to go to completion. 5. To determine the end point of the reaction, that is, the greatest dilution which stabilizes the gold against flocculation, visually identify the tube with the greatest dilution that retains a clear red color. To guard against possible errors in dilution, we use twice the amount of protein indicated by the test. If, for example, the dilution assay indicated that stabilization occurs at 1 : 16, then we would use 1 : 8 as the stabilizing dilution. If we want to prepare 100 ml of avidin-stabilized gold, we would add 2.5 ml of undiluted protein [volume desired/(dilution factor x volume of gold stabilized in the test, i.e., 5)].
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6. The protein is then added (concentration -< 1 mg/ml in 5 mM phosphate buffer) to a rapidly stirring solution of pH-adjusted gold sols. 7. After stirring for 15 min, a 5-ml sample is taken and tested for stability as in step 4. 8. The avidin-gold colloids are washed by centrifugation at 18,000 g for 3 hr at 4 °. This results in the formation of a loose pellet and a tight pellet. The supernatant fluid is discarded, and the loose pellet is resuspended to the original volume in 5 mM phosphate buffer (pH 7.5) and transferred to another centrifuge tube. The remaining tight pellet is discarded. 9. The gold colloids are repelleted by a second centrifugation as above. The resulting loose pellet is resuspended in sufficient 5 mM phosphate buffer (pH 7.5) such that its color approximates that of the initial solution, i.e., step 7 (above). The resultant avidin-gold colloidal suspension is transferred to a 100-ml beaker and stirred. While stirring, an equal volume of double-strength stabilizing buffer [18.0 g/liter NaCI, 12.1 g/liter Tris-HC1, 400 mg/liter polyethylene glycol (MW >20,000) (pH 7.5)] is added. 10. After 15 min of stirring, the avidin-gold colloids are placed in plastic tubes and stored at 4 ° until needed.18 The procedure described varies slightly from previous published methods. 7,19 It is important to note that we use the avidin-gold colloids within 1 month after production. Avidin-gold colloids older than 1 month have an increased tendency to bind nonspecifically to cells. Just before use, the avidin-gold colloids are absorbed against the cell line being studied. For example, 5 ml of avidin-gold colloids is absorbed against nearly confluent monolayers of mouse LM fibroblasts. Prior to absorption the LM cells are exhausted of exogenous biotin and cooled to 4°. Absorption is repeated 3 times for 30 rain at 4°. Large aggregates and debris are removed by centrifugation at 18,000 g for 20 min at 4 °.
Biotinyl Ligand-Avidin-Gold Method The procedure described is limited to in vitro conditions. This restriction is due to the significant concentrations of biotin present in the sera of 18 Since we usually u s e the a v i d i n - g o l d colloids within I m o n t h of production, we do not add azide or similar agents. In a n y case, for internalization studies (as well as for other studies in which viable cells are used), it is, o f course, not r e c o m m e n d e d to u s e a n y s u c h toxic antimicrobial agents. 19 R. E. Morris and C. B. Saelinger, Infect. Immun. 52, 445 (1986).
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animals. 2° This fact also dictates that, during the experimentation procedures, medium containing biotin or animal sera must not be used. Typically, we grow cells in complete medium with I0% sera. For 2 hr prior to the initiation of the experiment we exhaust the cells of exogenous biotin by incubation at 37° in HEPES-buffered Hanks' balanced salt solution (HBSS). Failure to include this step results in high levels of nonspecific binding by the avidin-gold colloids. The steps in the procedure are given below. 1. Wash all samples 3 times with cold HBSS; incubate at 4 ° for 15 min. 2. Add the biotinylated protein to the samples in a minimal volume of HBSS; incubate at 4 ° for 30 min. 21 3. Wash all samples 3 times with cold HBSS. 4. Add the avidin-gold conjugates in a minimal volume of HBSS to each sample; incubate for 30 min at 4 °. 5. To those samples which are to be warmed, add prewarmed HBSS and incubate on a warming plate. After appropriate incubation, wash samples 3 times with cold HBSS. 6. Wash all samples twice with cold 0.1 M sodium cacodylate buffer (pH 7.4) containing 0.05% CaC12 and 5.0% sucrose (SCB). 7. Fix all samples with SCB containing 2.0% paraformaldehyde and 2.5% (w/v) glutaraldehyde; fix for 30 min at 4 °. 8. Repeat step 6. 9. Post fix all samples with SCB containing 1% osmium tetroxide reduced with 1% potassium ferrocyanide22; fix for 1 hr at 4 °. 10. Repeat step 6. 11. Wash all samples twice with distilled water at 23°. 12. Begin dehydration by 3 washes with 70% (v/v) ethanol. 13. Stain e n b l o c for 10 min with 0.5% uranyl acetate in 70% (v/v) ethanol. 14. Continue dehydration by 2 washes with 70% ethanol followed by 3 washes with absolute ethanol. 15. Infiltrate and embed by standard procedures using epoxy resin. 2o R. E. Morris and C. B. Saelinger, Immunol. Today 5, 127 (1984). 21 C o n c e n t r a t i o n s and v o l u m e s of reagents used in these experiments vary from e x p e r i m e n t to experiment. F o r e x a m p l e , in one instance we m a y add l ml of a given biotinyl protein to a 16 × 85 m m Leighton tube; in a n o t h e r instance we m a y add 2.5 ml to a 100 m m petri dish. C o n c e n t r a t i o n s vary depending on the biotinyl ligand and the cell type. For most e x p e r i m e n t s we u s e L M cells grown on 16 x 85 m m glass Leighton tubes, using a l-ml v o l u m e of biotinyl toxin at concentrations ranging from 1 ng/ml to 1 p.g/ml. 22 M. J. K a r n o v s k y , J. Cell Biol. 51, 146a (1971).
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A control sample in which the biotinyl ligand is omitted should be included. This will measure nonspecific (background) binding of the gold colloid to cells. One of the advantages of the biotinyl ligand-avidin-gold system is that specific binding can be demonstrated by a competition assay. This is done by including a sample with a 100- to 200-fold excess of native ligand (not biotinylated) during the primary incubation step with the biotinyl ligand. The procedure described above is done on glass Leighton tubes. 23 Following polymerization at 60°, the resin containing the monolayer is freed from the glass by submersion in an ice bath. In preparation for sectioning, small areas of the plastic are drilled out with an electric cork borer (E. H. Sargent and Company, Birmingham, AL) and mounted on 3/4 × I/4 inch wooden dowels. 24 Alternatively, the cells can be grown on plastic petri dishes. At the conclusion of step 11, the cells are scraped from the plastic surface, placed in 1% low-temperature gelling agarose (Sigma), and pelleted (in a conical centrifuge tube) by low-speed centrifugation at 23 °. Following overnight solidification at 4°, the cell-rich regions of the agarose are diced into pieces of about 1 mm 3 and treated as described above. The samples are ultimately embedded in epoxy resin in BEEM capsules. The samples are viewed after preparation of ultrathin sections. The above procedure results in sufficient specimen contrast so that the samples do not require staining. Comments
We have used the biotinyl ligand-avidin-gold method to follow the intracellular trafficking of several different proteins. The majority of our experience has been with the intracellular trafficking of Pseudomonas exotoxin A (PE) by mouse LM fibroblasts. We have shown that LM cells, a cell line exquisitely sensitive to PE, internalize biotinyl-PE-avidin-gold by receptor-mediated endocytosis, route the complex to the Golgi region in endosomes, and ultimately deliver the complex to the lysosomal compartment. The entire process takes 20-30 min at 37°. 19,25,26We have also followed the internalization and intracellular trafficking of biotinyl diphtheria toxin by Vero cells, a cell line exquisitely sensitive to this toxin.
23 j. S. Sutton, Stain Technol. 40, 151 (1965). 24 R. E. Morris, G. M. Ciraolo, D. A. Cohen, and H. C. Bubel, In Vitro 16, 136 (1980). 2~ R. E. Morris, M. D. Manhart, and C. 13. Saelinger, Infect. Immun. 40, 806 (1983). 26 R. E. Morris, in "Microbiology 1985" (L. Leive, ed.), p. 91. American Society for Microbiology, Washington, D.C., 1985.
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The routing of diphtheria toxin in Vero cells is similar to that of PE in mouse LM cells. 27 Others have shown that ligands bound to gold are routed differently in cells than are native ligands. For example, van Deurs et alp showed that Vero and MCF-7 cells internalize native ricin by receptor-mediated endocytosis and that the ricin is routed by the endosomal system to the Golgi cisternae. In contrast, ricin-gold conjugates, although internalized by receptor-mediated endocytosis, are not observed in the Golgi. They estimated that each ricin-gold conjugate contains 2 to 4 ricin molecules per gold particle z8 and is therefore polyvalent. They further showed that monovalent conjugates, formed between ricin and horseradish peroxidase, are routed intracellularly as is native ricin. The authors concluded that the valency of the ligand conjugate dictates the intracellular trafficking pattern. Others have reported similar observations with transferringold conjugates. 1,2 Two experimental observations lead us to believe that the biotinyl ligand-avidin-gold technique circumvents this problem. First, biotinyl diphtheria toxin-avidin-gold complexes are routed differently in mouse LM cells than are biotinyl Pseudomonas toxin-avidin-gold complexes. Mouse LM cells are resistant to diphtheria toxin. The diphtheria toxingold conjugates are not internalized by receptor-mediated endocytosis and are not routed to the Golgi region. 29 This occurs in spite of the fact that LM cells have receptors for diphtheria toxin 3° and that diphtheria toxin and Pseudomonas toxin inhibit mammalian cell protein synthesis by identical mechanisms. We feel that this observation supports our hypothesis that the mechanism of entry and intracellular trafficking dictates the susceptibility of a cell to toxin. Second, our morphological observations are corroborated by biochemical data using native toxin. 31,32We feel that the biotinyl ligand-avidin-gold method allows for the ligand to be processed normally because the biotinyl ligand is permitted to interact with its receptor prior to addition of gold. Thus, the initial binding of ligand is not influenced by the gold, i.e., the valency is not altered. In conclusion, we advocate the use of the biotinyl ligand-avidin-gold technique as a tool to visualize intracellular trafficking. There are three 27 R. E. Morris, A. S. Gerstein, P. F. Bonventre, and C. B. Saelinger, Infect. Immun. 50, 721 (1985). z8 B. van Deurs, L. R. Pedersen, A. Sundan, S. Olsnes, and K. Sandvig, Exp. Cell Res. 159, 287 (1985). 29 R. E. Morris and C. B. Saelinger, Infect. Immun. 42, 812 (1983). 3o j. R. Didsbury, J. M. Moehring, and T. J. Moehring, Mol. Cell. Biol. 3, 1283 (1983). 3i C. B. Saelinger, R. E. Morris, and G. Foertsch, Eur. J. Clin. Microbiol. 4, 170 (1985). 3~ C. B. Saelinger and R. E. Morris, Antibiot. Chemother. (Basel) 39, 149 (1987).
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advantages of this technique over other methods for following intracellular trafficking at the ultrastructural level. First, because of the affinity of avidin for biotin, dissociation of the ligand from the electron-dense marker is negligible. Second, competition of native ligand and biotinyl ligand for a receptor on the cell surface can be measured. Third, the technique permits the use of fixation and embedding protocols which result in excellent ultrastructural preservation. This allows for more precise definition of intracellular locations.
[43] L o c a l i z a t i o n o f L e c t i n R e c e p t o r s
By RICHARD M. PINO Introduction The location and chemical composition of cell surface components have often been examined by ultrastructural cytochemical methods.~-5 A basic approach is the identification of monosaccharides using lectins. 6 With direct labeling, cells or tissues are treated with lectins that are coupled to ferritin, 7 which has inherent electron density. Lectins can also be coupled to horseradish peroxidase (HRP), 8 which can generate an electron-dense reaction product after incubation in a cytochemical medium. HRP methods are also applicable to light microscopic cytochemistry analysis. The avidin-biotin method 9 is a reliable indirect method that can be used for lectin affinity 1°,1~ and immunocytochemical 4,1°,12 localizations on i D. Danon, L. Goldstein, Y. Marikovsky, and E. Skutelsky, J. UItrastruct. Res. 38, 500 (1972). 2 p. p. H. DeBruyn and S. Michelson, J. Cell Biol. 82, 708 (1979). 3 E. Essner, R. M. Pino, and R. A. Griewski, Curr. Eye Res. 1, 381 (1981). 4 R. M. Pino, Invest. Ophthalmol. Visual Sci. 27, 840 (1986). 5 R. M. Pino, Cell Tissue Res. 250, 257 (1987). 6 N. Sharon and H. Lis, Science 177, 949 (1972). 7 G. L. Nicolson and S. J. Singer, J. CellBiol. 60, 236 (1974). 8 W. Bernhard and S. Avrameas, Exp. Cell Res. 64, 232 (1971). 9 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). l0 E. A. Bayer, M. Wilchek, and E. Skutelsky, FEBS Left. 68, 240 (1976). H S. M. Hsu and L. Raine, J. Histochem. Cytochem. 30~ 157 (1982). 12 S. M. Hsu, L. Raine, and H. Fanger, J. Histochern. Cytochem. 29, 577 (1981).
METHODS IN ENZYMOLOGY, VOL. 184
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