ARTICLE |
Correspondence to: Toshihiro Takizawa, Dept. of Anatomy, Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Tochigi 329-04, Japan.
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Summary |
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We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphatase activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes. (J Histochem Cytochem 46:11-17, 1998)
Key Words: freeze-fracture electron, microscopy, freeze replica, octyl-glucoside, immunocytochemistry, enzyme cytochemistry, HLA class I, CD16, CD62L, alkaline phosphatase, neutrophils
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Introduction |
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Freeze-fracture cytochemistry is a method that combines freeze-fracture electron microscopy with cytochemistry. It reveals the characteristics of biological membranes by labeling membrane constituents at high resolution and extensive fields of freeze-fractured membranes with cytochemical probes. In this technique, immunocytochemistry using immunogold probes has been applied widely to freeze-fracture cytochemistry (i.e., freeze-fracture immunocytochemistry). It has been a powerful technique for revealing the surface topology in biological membranes (e.g.,
We introduce here octyl-glucoside (OG) digestion for this new double labeling method. Our results show that immunocytochemistry and enzyme cytochemistry can be combined to demonstrate the localization of two different proteins on the same replicas. The method described here should serve as a unique additional approach for the study of topology and dynamics of many molecules in biological membranes.
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Materials and Methods |
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Reagents
The following chemicals were purchased from Sigma (St Louis, MO): cerium chloride, dextran, dimethyl sulfoxide (DMSO), ß-glycerophosphate, Hank's balanced salt solution, Histopaque 1083, levamisole, Tricine, and N-tris methyl-3-aminopropanesulfonic acid (TAPS). Glutaraldehyde (25% aqueous), maleic acid, 1-O-n-octyl-ß-D-glucopyranoside (octyl glucoside, OG) and sucrose were obtained from Nacalai Tesque (Kyoto, Japan). Sodium dodecylsulfate (SDS) was supplied by Polyscience (Warrington, PA). Sodium cacodylate was obtained from Wako (Osaka, Japan). All other chemicals were of the highest grade available.
Mouse monoclonal anti-human leukocyte antigen (HLA) Class I (clone W6/32) and mouse monoclonal anti-human CD16 (Fc -receptor III) (clone DJ130c) were obtained from Dako (Glostrup, Denmark). Mouse monoclonal anti-human CD62L (L-selectin) (clone FMC46) was from Novocastra (Newcastle upon Tyne, UK). Mouse monoclonal anti-human CD16 (clone gran 1) was kindly provided by Dr. Clark L. Anderson (Ohio State University). Ten-nm colloidal gold was prepared by the tannic acid/citrate method and then conjugated with affinity-purified goat anti-mouse IgG (Cappel; Durham, NC) (
Cell Isolation
Whole human blood was collected from healthy adult men after obtaining informed consent. Neutrophils were purified from whole blood in the unstimulated state, as described previously (
Cytochemistry on Replicas
Unstimulated human neutrophils were rapidly frozen and fractured as reported previously (
After OG digestion, the replicas were rinsed with three changes of PBS and then incubated with the following antibodies for 60 min at room temperature (RT): anti-HLA Class I (33.0 µg/ml), anti-CD16 (clone: gran 1, 5 µg/ml; DJ130c, 2.7-5.4 µg/ml), anti-CD62L (diluted 1:10; this dilution was of the material supplied by the manufacturer). The replicas were then rinsed in PBS three times over 15 min. Detection of the primary antibody binding sites was achieved with a 60-min incubation in goat anti-mouse IgG conjugated with 10-nm gold. All antibody solutions were diluted with PBS containing 10% normal goat serum. The replicas were rinsed three times in PBS and then transferred to 0.1 M cacodylate buffer (pH 7.4) containing 5% sucrose.
Incubation for the detection of alkaline phosphatase (ALPase) activity was subsequently performed for 60 min at 37C with constant agitation. This medium contained 50 mM Tricine, 100 mM TAPS, 2 mM CeCl3, 5 mM ß-glycerophosphate, 5 mM MgSO4, and 5% sucrose, a modification of the medium of
After the cytochemical reactions, the replicas were postdigested in PBS containing 20 mM OG, overnight at RT. The samples were then fixed in 2% glutaraldehyde in cacodylate buffer for 10 min at RT, washed with distilled water, collected on Formvar-coated copper grids, and examined with a Hitachi H-7000 electron microscope operated at 100 kV. During the entire procedure the replicas were floated on the various liquids employed.
Control immunocytochemical and enzyme cytochemical incubations consisted of omission of the primary antibodies from the immunoreaction mixture and inclusion of the alkaline phosphatase inhibitor levamisole (2.5 mM) in the complete enzyme reaction mixture, respectively.
In parallel experiments, we tested the possibility of using SDS, as reported by
Morphometric Analysis of Immunogold in Human Neutrophils
The labeling density of 10-nm colloidal gold-IgG particles indicating the localization of HLA Class I was determined. Negatives of electron micrographs of the replicas were printed at the same magnification (x 63,000). The area of the exoplasmic halves (E-faces) of plasma membranes was measured and the number of individual immunogold particles with the E-faces was counted on the micrographs.
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Results |
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The replica digestion before the cytochemical reactions was crucial for this new method. Experiments were carried out with various concentrations of OG in the replica digestion solution with the replicas. For immunocytochemical fracture-labeling, OG not only efficiently digested unfractured cell components but also adequately retained immunocytochemical antigenicity on the split membranes stabilized with Pt/C. OG at 0.6-60 mM gave good results for the antigens tested. For example, HLA Class I was detected primarily on freeze-fractured plasma membranes in resting human neutrophils (Figure 1A). For enzyme cytochemical fracture-labeling, OG was able to preserve enzyme activity on the replicas. ALPase activity was demonstrated in association with the freeze-fractured membranes of small intracellular granules (Figure 1B). Best results were found when OG concentrations of 6-20 mM were included in the digestion medium. Higher concentrations of OG (more than 20 mM) led to failure in detection of the reaction product for ALPase activity (Figure 2). In addition, lower concentrations of OG (less than 6 mM) led to poorer dissolution of unfractured cell components attached to the replicas. Such was the case even though intracellular enzyme cytochemical staining for ALPase was detectable. Optimal conditions for the use of OG in this combined cytochemical approach were found when the replicas were digested in 6-20 mM OG-PBS solution for 1 hr at 4C. In these experiments the detection of enzyme activity on replicas was more sensitive to the OG digestion than was detection of antigens (Figure 2). After SDS digestion, it was possible to detect antigens on replicas. However, the enzyme cytochemical results obtained in this case were not acceptable because ALPase activity was not detected (Figure 1C).
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We then combined the immunocytochemical localization of HLA Class I with the enzyme cytochemical localization of ALPase activity on the same replica. The former was found on the plasma membranes of human neutrophils and the latter in the small cytoplasmic granules (Figure 3A). The immunogold particles showing the localization of HLA Class I were restricted to the E-faces of the plasma membranes (Figure 1A and Figure 3A). The cerium reaction product demonstrating ALPase activity was associated predominately with the E-faces of the intracellular granules (Figure 1B and Figure 3A).
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Localization of CD62L and ALPase in the same cells was carried out by this new fracture-labeling method. In unstimulated cells, CD62L as well as HLA Class I was demonstrated with 10-nm colloidal gold and was localized on the E-faces of the plasma membranes. ALPase was detected in the cytoplasmic organelles, as mentioned above (Figure 3B). This is essentially consistent with earlier findings suggesting that in unstimulated human neutrophils, L-selectin is constitutively expressed on the cell surface and ALPase is stored in a intracellular compartment (e.g.,
Double labeling of CD16 and ALPase was also achieved on the same replicas (Figure 3C). In unstimulated cells, CD16 was present both on the E-faces of the plasma membranes and the E-faces of cytoplasmic granules. Immunogold particles were sometimes present on the E-faces of the granules that were labeled with the reaction product demonstrating ALPase (Figure 3C). This result suggests heterogeneity in this population of granules (manuscript in preparation). It should be noted that because the electron density of immunogold particles was distinct from that of the cerium phosphate reaction product, co-localization of two different proteins labeled with different cytochemical probes was easily recognized.
Control immunocytochemical incubations consisted of omission of the primary antibodies. Controls for the enzyme cytochemical reaction consisted of adding the ALPase inhibitor levamisole to the complete medium. In each control situation the appropriate labeling was absent (Figure 3D).
Occasionally unfractured cell components were incompletely lysed in the digestion medium and remained attached to the cytochemically labeled replica. These remnants on the replica not only made observation of cell ultrastructure difficult but also induced high background due to the superimposition of the cytochemical probes involved in them on the replica membrane (data not shown). Therefore, the postdigestion after the cytochemical reactions was of importance to overcome this obstacle. We tested many cleaning agents (e.g., OG, SDS, sodium hypochlorite, sodium hydroxide, hydrochloric acid). OG yielded superior results for the postdigestion over any other compound tested.
In this method and in the combined method for cryosections (
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Discussion |
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Freeze-fracture immunocytochemistry was first introduced as the "fracture-label" technique for carbohydrate topology in biological membranes (
The combination of immunocytochemistry and enzyme cytochemistry is also desirable in certain experimental situations. In our studies, we have used human neutrophils as a model system to investigate the possibility that immunocytochemistry and enzyme cytochemistry could be combined in freeze-fracture cytochemistry. We initially tested the feasibility of SDS-FRL for the double labeling on the same replicas with different cytochemical probes. However, we found that SDS-FRL caused significant enzyme activity loss from Pt/C-stabilized membrane halves (see Figure 1C). It could be speculated that SDS, a strong ionic detergent, completely denatures native enzyme molecule structures, thereby rendering them inactive and unusable for enzyme cytochemical studies. Therefore, in place of SDS, we sought a more suitable compound for replica digestion for this new method. In previous studies, we introduced an enzyme cytochemical fracture-labeling method. ALPase activity was demonstrated on replicas by the use of Triton X-100 or saponin digestion and after ultrasonication treatment (
OG, a nonionic detergent, was initially reported as an effective agent for solubilizing membrane proteins (
The order of the reactions is important. The immunocytochemical localization is always the first step, to eliminate the probability of interference from the enzyme cytochemical reactions. The enzyme cytochemical reaction may modify the efficiency of the subsequent immunolocalization. This might be a particular problem when co-localization of the enzyme cytochemical and immunocytochemical products occurs. The metal precipitations from enzyme cytochemical reaction may cover antigenic sites or in some other ways inhibit antigen-antibody binding. In the present study, immunocytochemistry followed by enzyme cytochemistry showed the co-localization of CD16 and ALPase on the same intracellular granule (see Figure 3C). Therefore, we have developed a procedure in which combined immunocytochemistry and enzyme cytochemistry can be conducted without these potential complications.
In our experiments, it was still possible that the subsequent cytochemical reaction and the postdigestion might lead to loss of immunolabeling from the replicas. This was tested by morphometric analysis of the immunogold labeling and was found not to occur (see Figure 4). In other applications of this combined approach, similar control experiments would be necessary to determine whether or not the particular enzyme cytochemical reaction employed affects immunocytochemical labeling. In addition, the enzyme cytochemical reaction product (i.e., cerium phosphate) is not affected by OG digestion. Indeed, it is stable after treatment with household bleach, one of the most powerful replica cleaning agents (
Combined immunocytochemical and enzyme cytochemical reactions on the same replicas would have many applications and should provide new and important information that may be difficult to obtain by other approaches, e.g., the localization of adhesion molecules and glycosyl-phosphatidylinositol (GPI)-anchored proteins in human neutrophils. It has been recently suggested that adhesion molecules (e.g., CD11b, CD62L) and ALPase, which is a GPI-anchored protein, simultaneously change these subcellular localizations and surface expressions in human neutrophils during stimulation (
In conclusion, we show a new freeze-fracture cytochemistry combining immunocytochemistry and enzyme cytochemistry. This technique enables us to reveal the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e. immunogold particles and cerium phosphate reaction product). This technique should be a useful addition for studying the ultrastructural organization of biomembranes.
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Acknowledgments |
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Supported by grants-in-aid for Scientific Research (nos. 08670030 and 09770016) from the Ministry of Education, Science, Sports and Culture of Japan (TS, TT), by a grant from the Kazato Research Foundation (TT), and by a grant from the Nippon Foundation (TT).
We thank Dr Clark L. Anderson for his generous gift of antibody. We are deeply indebted to Ms Kiyomi Inose, Ms Kaori Ishikawa, Ms Chiaki Ishijima, and Ms Megumi Yatabe for excellent technical assistance.
Received for publication August 18, 1997; accepted August 25, 1997.
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Literature Cited |
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Andersson Forsman C, Pinto da Silva P (1988) Fracture-flip: new high-resolution images of cell surfaces after carbon stabilization of freeze-fractured membranes. J Cell Sci 90:531-541[Abstract]
Baron C, Thompson TE (1975) Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine. Biochim Biophys Acta 382:276-285[Medline]
Borregaard N, Kjeldsen L, Sengeløv H, Diamond MS, Springer TA, Anderson HC, Kishimoto TK, Bainton DF (1994) Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators. J Leukocyte Biol 56:80-87[Abstract]
Cain TJ, Liu Y, Takizawa T, Robinson JM (1995) Solubilization of glycosyl-phosphatidylinositol-anchored proteins in quiescent and stimulated neutrophils. Biochim Biophys Acta 1235:69-78[Medline]
Fujimoto K (1995) Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins: application to the immunogold labeling of intercellular junctional complexes. J Cell Sci 108:3443-3449
Fujimoto K, Møller JV, Maunsbach AB (1996a) Epitope topology of Na, K-ATPase subunit analyzed in basolateral cell membranes of rat kidney tubules. FEBS Lett 395:29-32[Medline]
Fujimoto K, Pinto da Silva P (1989) Surface views of nuclear pores in isolated rat liver nuclei as revealed by fracture-flip/Triton-X. Eur J Cell Biol 50:390-397[Medline]
Fujimoto K, Umeda M, Fujimoto T (1996b) Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling. J Cell Sci 109:2453-2460
Gould RJ, Ginsberg BH, Spector AA (1981) Effect of octyl ß-glucoside on insulin binding to solubilized membrane receptors. Biochemistry 20:6776-6781[Medline]
Kobayashi T, Robinson JM (1991) A novel intracellular compartment with unusual secretory properties in human neutrophils. J Cell Biol 113:743-756[Abstract]
Helenius A, Simons K (1975) Solubilization of membranes by detergents. Biochim Biophys Acta 415:29-79[Medline]
Pinto da Silva P (1987) Topology, dynamics, and molecular cytochemistry of integral membrane proteins: a freeze-fracture view. In Harris JR, Horne RW, eds. Electron Microscopy of Proteins. Vol 6. London, Academic Press, 1-38
Pinto da Silva P, Kachar B, Torrisi MR, Brown C, Parkison C (1981a) Freeze-fracture cytochemistry: replicas of critical point-dried cells and tissues after fracture-label. Science 213:230-233[Medline]
Pinto da Silva P, Kan FWK (1984) Label-fracture: a method for high resolution labeling of cell surfaces. J Cell Biol 99:1156-1161[Abstract]
Pinto da Silva P, Parkison C, Dwyer N (1981b) Freeze-fracture cytochemistry: thin sections of cells and tissues after labeling of fracture faces. J Histochem Cytochem 29:917-928[Abstract]
Pinto da Silva P, Parkison C, Dwyer N (1981c) Fracture-label: cytochemistry of freeze-fracture faces in the erythrocyte membrane. Proc Natl Acad Sci USA 78:343-347[Abstract]
Ru-Long S, Pinto da Silva P (1990) Simulcast: contiguous views of fracture faces and membrane surfaces in a single cell. Eur J Cell Biol 53:122-130[Medline]
Shinoda K, Yamaguchi T, Hori R (1961) The surface tension and the critical micelle concentration in aqueous solution of ß-D-alkyl glucosides and their mixtures. Bull Chem Soc Japan 34:237-241
Slot JW, Geuze HJ (1985) A new method of preparing gold probes for multi-labeling cytochemistry. Eur J Cell Biol 38:87-93[Medline]
Takizawa T, Nakazawa E, Saito T (1997a) Freeze-fracture enzyme cytochemistry reveals the distribution of enzymes in biological membranes: enzyme cytochemical label-fracture and fracture-label. Acta Histochem Cytochem 30:77-84
Takizawa T, Robinson JM (1993) Combined immunocytochemistry and enzyme cytochemistry on ultra-thin cryosections: a new method. J Histochem Cytochem 41:1635-1639
Takizawa T, Saito T (1996) Freeze-fracture enzyme cytochemistry: application of enzyme cytochemistry to freeze-fracture cytochemistry. J Electron Microsc 45:242-246[Medline]
Takizawa T, Saito T (1997b) New fracture-labelling method: alkaline phosphatase in unstimulated human neutrophils. J Electron Microsc 46:85-91[Abstract]