Immunostaining of Glycosaminoglycans and Proteoglycans in ... · Immunostaining of Glycosaminoglycans and Proteoglycans in Marine ... 2Laboratório de Bioquímica e Biologia Celular

Immunostaining of Glycosaminoglycans and Proteoglycans in Marine Organisms

C. Monteiro de Barros1, M. S. Gonçalves Pavão2 and S. Allodi3

1Laboratório Integrado de Morfologia, Núcleo em Ecologia e Desenvolvimento Sócio-ambiental de Macaé – NUPEM, Campus Macaé, Universidade Federal do Rio de Janeiro, Brazil.

2Laboratório de Bioquímica e Biologia Celular de Glicoconjugados, Instituto de Bioquímica Médica and Hospital Universitário Clementino Fraga Filho – HUCFF, Universidade Federal do Rio de Janeiro, Brazil.

3Laboratório de Neurobiologia Comparativa e do Desenvolvimento, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil.

Immunocytochemistry is the specific use of labeled antibodies to locate antigens in tissue sections, as revealed by fluorescent dyes, enzymes, or colloidal gold. The method has advantages over traditionally used enzyme-staining techniques, since antibodies allow highly specific binding to single sequences of amino acids in proteins or disaccharides in glycosaminoglycans. Carbohydrates are the most abundant and structurally diverse organic molecules in nature. The location of carbohydrates at the forefront of the cell surface enables them to interact effectively with the immune system, and their role as a cell antigenic determinant has been firmly established and termed glycoimmunology. The antigenic mechanism of carbohydrate antigens is somewhat similar to that of proteins. Generally, antibodies recognize intact glycosaminolgycan chains or specific epitopes generated by lyases. Antibodies against specific peptide sequences in a proteoglycan or glycoprotein are also frequently used; however, the technique is poorly explained in the literature. Therefore, in this chapter we discuss techniques used in both light and electron microscopy to identify glycosaminoglycans. Protocols for fixation, dehydration, and embedding for specific recognized glycosaminoglycans in marine invertebrates are also discussed.

Keywords: Glycosaminoglycans, proteoglycans, invertebrates, immunohistochemistry, glycoimmunology

1. Introduction

Immunocytochemistry is the specific use of labeled antibodies to locate antigens in tissue sections, as revealed by fluorescent dyes, enzymes, or colloidal gold. The method has advantages over traditionally used enzyme-staining techniques, since antibodies allow highly specific binding to single sequences of amino acids in proteins, disaccharides in glycosaminoglycans (GAGs), and disaccharides or amino-acid sequences in proteoglycans [1]. Immunocytochemical techniques originated with the work of Albert H. Coons and colleagues (1955), who first labeled an antibody with fluorescent dye and used it to identify antigens in tissue sections. However, the term “antibody” was only employed about 30 years later by Paul Ehrlich in his article “Experimental Studies on Immunity”, published in October 1991 [2]. Immunohistochemical methods have been widely used by scientists to locate specific amino-acid sequences in tissues. However, the use of antibodies to locate GAGs and proteoglycans is still limited. In this chapter, we discuss immunohistochemistry protocols that are currently used to detect GAGs and proteoglycans in tissue sections and to determine the ultrastructural locations of these macromolecules in cells from marine organisms.

2. General Considerations on Proteoglycans and GAGs

Proteoglycans consist of a core protein to which one or more GAG chains are covalently linked. GAGs consist of repeating disaccharide units composed of an N-acetylated or N-sulfated hexosamine and either an uronic acid (glucuronic acid or iduronic acis) or galactose. With the exception of hyaluronic acid, all GAGs contain sulfate groups at specific carbons on the hexosamine and/or uronic acid. Virtually all animal cells contain proteoglycans that are secreted into the extracellular matrix, inserted into the plasma membrane, or stored in secretory granules [3]. Mammalian matrix proteoglycans include small interstitial proteoglycans (decorin, biglycan, fibromodulin), a proteoglycan form of type IX collagen, members of the agrecan family of proteoglycans (agrecan, brevican, neurocan, or versican). Some of these proteoglycans contain only one GAG chain (e.g., decorin), whereas others have more than 100 chains (e.g., agrecan). Matrix proteoglycans typically contain GAGs of the chondroitin and dermatan sulfate type. However, heparan sulfate-containing proteoglycans from the perlecan and agrin families abound in basement membranes. In addition, heparin proteoglycans (serglycin) are found in intracellular granules of immune cells [4]. Several reports have revealed the great structural diversity of vertebrate [5,6,7] and invertebrate GAGs [8,9,10]. From primitive invertebrates to more-recent vertebrate chordates, the structure of these molecules has been shown to be extremely heterogeneous. Different sulfation patterns are related to specific organs and tissues. Additionally, complex GAGs may be expressed differently during development. Therefore, immunohistochemistry is a valuable tool to understand how structure relates to specific organs and tissues, and to the specific stage of development [11].

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

© 2012 FORMATEX 458

2.1. Structure of GAGs:

Heparin (Hep) and heparan sulfate (HS): The most common disaccharide unit is composed of GlcNAc 1,4-linked to GlcA. The structure of Hep and HS is very similar; however, heparin contains more N-sulfate groups than N-acetyl groups, and the concentration of O-sulfate groups exceeds that of N-sulfate [4,12]. Chondroitin sulfate (CS): Consists of repeating units of sulfated GlcA-GalNAc disaccharides, polymerized into long chains that can be easily identified using bacterial chondroitin lyases (chondroitinases). In vertebrates, sulfation in CS is very complex, involving several sulfotransferases that add sulfate groups at carbon 4 or 6 on the GalNAc residues, and also at carbon 2 on the IdoA residues in dermatan sulphate (DS).

Table 1: Different types of chondroitin sulfates.

Chondroitin sulfate type Disaccharide repeat Systematic name

Chondroitin sulfate A GlcAβ1-3GalNAc4S Chondroitin-4-sulfate

Chondroitin sulfate B IdoAα1-3GalNAc4S Chondroitin-6-sulfate

Chondroitin sulfate C GlcA2Sβ1-3GalNAc6S Chondroitin-6-sulfate

Chondroitin sulfate D GlcAβ1-3GalNAc6S Chondroitin-2,6-sulfate

Chondroitin sulfate E GlcAβ1-3GalNAc4,6diS Chondroitin-4-6-sulfate

Dermatan sulfate (DS): This galactosaminoglycan is composed of alternating disaccharide units of hexuronic acid (IdoA or GlcA) 1,3 linked to GalNAc. Variations in the degree of sulfation on both hexuronic acid (2-O-sulfated) and GalNAc (4-O- or/and 6-O-sulfated) are responsible for the extensive heterogeneity of this polymer [6]. Differently from CS, DS refers to a glycan that contains one or more IdoA disaccharides. It is also referred to as CS B, although it is no longer classified as a form of CS [4]. Keratan sulfate (KS): Keratan is a sulfated polylactosamine chain and contains a mixture of nonsulfated, monosulfated and disulfated disaccharides. The basic repeating disaccharide unit is →3Galβ-1→4GlcNAc-β→1. It can be sulfated at carbon 6 of both Gal and GlcNAc monosaccharides. Bacterial keratanase and chondroitinase ABC degrade KS at specific positions [13]. Hyaluronic acid (HA): This is composed of D-GlcA and D-N-GlcNac, linked via alternating β-1→4 and β-1→3 glycosidic bonds. Several types of hyaluronidases, enzymes that degrade HA, are known to generate either tetrasaccharides or disaccharides as end products [4].

3. Antibodies

Antibodies or immunoglobulins (Igs) are glycoproteins of the immune system that identify and neutralize foreign substances in the body. Ig molecules are heterodimers composed of four polypeptide chains, two identical heavy chains of about 50-60 kDa, and two small light chains of about 23 kDa. The heavy and light chains are linked by interchain disulfide bridges. This prototype structure is common for all monomeric Ig molecules. Polymeric Ig of higher molecular weight is formed by 2 to 6 four-chain subunits, similar to the monomeric Ig molecule. They possess one or two additional peptide chains that are important for the formation and stabilization of Ig polymers [9]. The Ig molecules are found at the surface of B-lymphocytes or soluble in the blood and lymph. In mammals, antibodies are divided into five classes, IgG, IgM, IgA, IgE, and IgD, based on the numbers of prototype structures and the type of heavy-chain polypeptides [14].

3.1 Production of antibodies

Antibodies are produced by immunization of suitable animals or by production of hybridomas, using myeloma cells (plasma cell tumor) and activated B-lymphocytes from mouse spleen. However, the antibodies produced by these two methods are different. An animal injected with an antigen generates multiple antibodies against different epitopes, whereas the hybridome produces an antibody against a single epitope. A single clone of B-lymphocytes produces antibodies against only a single specific epitope. Once the B-lymphocyte starts to produce a single type of antibody, it divides and gives rise to many B-cells, all producing antibodies against a specific epitope. This is called a lymphocyte B-cell clone. The ability to induce the production of antibodies varies among different molecules. Even different parts of the same molecules are better antigens than others [15].



3.1.1 Production of antibodies against GAGs and proteoglycan epitopes

Carbohydrates are the most abundant and structurally diverse organic molecules in nature. The location of carbohydrates on the outer cell surface enables them to effectively interact with the immune system, and their role as cell antigenic determinants has been firmly established and its study termed glycoimmunology [16]. As a general rule, low-molecular-weight carbohydrates are not immunogenic unless coupled to an immunogenic carrier, a hapten. The antigenic mechanism of carbohydrate antigens is somewhat similar to that of proteins. In general, glycans activate B-lymphocyte cells in a thymus-independent type-2 response. Because carbohydrates typically stimulate B-cells in a T-cell independent manner, IgM is the predominant immunoglobulin produced. In addition, affinity maturation and development of memory cells are barely present [15,16].

3.2 Types of GAG and proteoglycan antibodies

Antibodies that recognize intact GAG chains or specific epitopes generated by GAG-lyases are commercially available. Generally, anti-GAG monoclonal antibodies are produced in the mouse, against intact human GAG chains, and these antibodies can cross-react with homologous epitopes from other species such as monkey, cat, rat and mouse, but not with other types of GAG [17]. Some of these antibodies identify the intact GAG chains, but others can only identify a disaccharide sequence or “stub” of GAG. The “stub” antibodies can recognize a specific GAG after extensive digestion using a defined enzyme. Usually, the antibody specificity is according either to the sulfated position (e.g., 4S, 6S) or unsulfated position (OS) [17]. This is because the self-association among the GAGs and their interaction with other components of the extracellular matrix may be affected by the particular charge on the GAG molecule. Therefore, these antibodies provide an important tool to study the distribution of specific types of some GAG chains that appear to show significant tissue specificity. The most common “stub” antibodies against GAGs are: Anti-proteoglycan ΔDi-4S, anti-proteoglycan ΔDi-6S, and anti-proteoglycan ΔDi-OS [17].

3.3 Antibody Labeling

In order to observe an immunoreaction under the microscope, the antibody must be labeled. The most frequent labeling methods make use of an enzyme or a fluorophore. Colloidal gold or biotin can also be used. The enzyme-labeled antibody can be revealed by histochemical methods via chromogenic substrate reactions. In a fluorophore-labeled immunoreaction, the antibody is detected directly under a fluorescent microscope. Electron-dense labels such as colloidal gold are visible under the electron microscope without any additional treatment. A biotin label can be exploited under light, fluorescence, or electron microscopy in combination with the Avidin-Biotin Complex (ABC) technique. Similarly to biotin, some other haptens such as digoxigenin (DIG) or denitrophenol (DNP) can also be coupled to antibodies [18].

4. Immunohistochemistry Methods

Immunohistochemistry methods can be divided into two main groups: direct or indirect. In general, depending on the molecule that is conjugated to the antibody, the reactions can be observed under light, fluorescent, or electron microscopy.

4.1 Direct, Indirect, and Avidin-Biotin Methods

The conjugated molecule is bound to the antibody against the molecule of interest (primary antibody) [19]. This method is inflexible, and may make labeling difficult when the use of commercially labeled direct conjugates is impractical. In the indirect method, the primary antibody is unlabeled and a second anti-immunoglobulin antibody directed to the constant portion of the first antibody, called a secondary antibody, is tagged to the marker. Note that the secondary antibody must be directed to the isotype from which the primary antibody is made. For example, if the primary antibody is an IgM, particularly if it is a monoclonal antibody, then the second antibody must be an anti-IgM antibody. Anti-IgG alone will not react with an IgM primary antibody. The avidin-biotin method has greater sensitivity than the other two methods mentioned above, due to the amount of label that is eventually bound to the antigenic site. Avidin is a large glycoprotein extracted from egg white (albumen). It has four binding sites per molecule, for a low-molecular-weight vitamin called biotin, and a particularly high affinity for it. Biotin is a vitamin found in several tissues and can be extracted from egg yolk. Each biotin molecule has one binding site for avidin, and can be attached through other sites either to an antibody or to any other macromolecule, such as an enzyme, fluorophore or other label. Avidin can also be labeled, and therefore these reagents can be used in a variety of immunostaining techniques. The increased sensitivity results from the large number of biotins that can be attached to an antibody [20]. The indirect and the avidin-biotin methods are most often used.



5. Immunohistochemistry for observation at the light-microscopy level

Biological samples must be embedded in a hard matrix, to allow sufficiently thin sections to be cut; for light microscopy, the sections are generally 5-7 μm thick. Paraffin wax is the most frequently used embedding medium, although other plastic formulations are also used. Every step can affect the final result. This technique is simpler than those used to prepare samples for electron microscopy, and the reagents are less expensive. In order to obtain the best results, care must be taken during the processing steps. For the immunoreactions, the sections obtained must be blocked for unspecific sites, permeabilized, incubated with the primary antibody, washed, incubated with the secondary antibody, washed again, and mounted before examination under the light or fluorescence microscope (immunofluorescence), depending on the secondary antibody tag [21].

5.1 Fixation

The goal of fixation is to preserve all components of a tissue sample, with minimum alteration from the living state. Additionally, fixation should protect tissues (or only cells) against disruption during embedding and sectioning. Fixatives always fall short of these ideals [22]. Fixation must be started as soon as possible after the tissue sample is obtained. Perfusion fixation is recommended, if possible, as it allows the fixative to penetrate the tissue thoroughly and rapidly. For immersion, the tissue sample must be small. Also, it is very important that for immunoreactions, the fixative used prevent damage to the antigen [21,23]. In general, paraformaldehyde (PFA) is the fixative of choice for observing the reaction under the light microscope, since it gives the best results. For GAGs, one of the fixatives described below should be used . All of them give good results [24,25].

5.1.2 Types of Fixation

Fixation may be achieved by using a solution of chemicals, the form most commonly employed, or it may be physical, such as heat denaturation, freezing, or air drying. Generally, in order to fix GAGs or proteoglycans, chemical fixation can be used with good results. However, we recommend chemical fixation, which is explained in more detail below [26].

5.1.2.1 Chemical Fixation

It is produced using substances that react with specific sites on the molecules. Chemical fixatives can denature proteins to different degrees, depending on the molecular structure of the fixative agent. The penetration modifies the polypeptide chain, which exposes hydrophobic radicals on the surface: this phenomenon is called coagulation, and the proteins form a precipitate [27].

5.1.2.1.1 Formaldehyde

Formaldehyde is often recommended as a fixative for immunohistochemistry. In general, PFA is used, which is a formaldehyde polymer without methanol contamination. In immunohistochemical reactions, this compound is reported to exhibit few cross-combinations, and for this reason, it preserves the immunogenic properties. A combination of glutaraldehyde (GLA) and formaldehyde is sometimes used, particularly for electron microscopy [28,29]. Formaldehyde is thought to cause cross-linking of the peptide chain. The reactive functional groups identified so far include amino, amide, guanidine, thiol, phenol, imidazol, and indol. Formaldehyde does not preserve soluble polysaccharides, but prevents extraction of glycogen, provided the duration of fixation is not too long. GAGs are not preserved by formaldehyde unless they are bound to proteins [30].

5.2 Marine invertebrates

5.2.1 Fixation

In general, fixation of marine-tissues gives less satisfactory results than for tissues of other invertebrates or mammals. Although GLA for pre-embedding immunohistochemistry is used to preserve proteoglycans or GAGs, some modifications in the regular protocol are based on specific particularities. Since tissues of marine organisms are isosmotic with seawater, all fixative mixtures must be prepared with filtered seawater. For light microscopy, the best results are obtained with phosphate buffer prepared in seawater with 4% PFA [29].

5.2.2 Washing, dehydration, and clearing

After the sample is fixed, the next step is washing the tissue. Washing is the simplest means of reversing the effects of fixative solutions. If the tissue is to be embedded in paraffin or plastic, all traces of water must be removed, because water and paraffin or plastic are not miscible. The dehydration process is accomplished by passing the tissue through a



series of increasing ethanol concentrations. The tissue pieces are transferred sequentially to 30%, 50%, 70%, 80%, 90% and 100% ethanol solutions for a few minutes each, depending on the size of the sample. The sample should be immersed twice in 100% ethanol to ensure that no water is left within the tissue [31]. After dehydration, the tissue to be embedded in paraffin must be cleared. Clearing is achieved using an intermediate fluid that is miscible with both ethanol and paraffin, since these two compounds are also not miscible. For paraffin embedding, benzene, toluene, chloroform and xylene are the most commonly used clearing agents. Most of them are harmful, and therefore must be treated with care. The most commonly used clearing agent is xylene. For this step, the tissue is immersed in 50:50 v/v ethanol/xylene for a few minutes, then in pure xylene, next in a mixture of xylene and paraffin (50:50 v/v), and finally in pure liquid paraffin, i.e., at 56-58º C (the melting temperature of paraffin) [32].

5.2.3 Embedding and cutting

After the steps described above, the tissues can be embedded in paraffin, nitrocellulose, or various formulations of plastics. Paraffin is the least expensive and therefore the most commonly used material. Recently, plastics have become more frequently used, primarily because they allow thinner sections and also because the sectioning is easier than with paraffin. After infiltration, the tissue is placed in the embedding mold with liquid paraffin and then cooled. Having infiltrated the tissue, the paraffin becomes solid, forming a block that can be sectioned [31,32].

5.2.4 Antigen retrieval

The process now known as antigen retrieval is applied to aldehyde-fixed tissues in which antigenicity has been reduced by the formation of hydroxyl-methylene bridges between components of the amino-acid chains of proteins. In many instances, immunoreactivity can be restored without compromising the structure of the tissues [33].

5.2.4.1 Heat-mediated antigen retrieval

A major step forward in immunohistochemistry was made with the discovery that some antigens previously unreactive in formalin-fixed, paraffin-embedded tissue, even after protease treatment, could be retrieved by heating the sections in a solution of a heavy-metal salt in a microwave oven, without deleterious effects on the tissue structure [34]. Subsequently, it was shown that the rather toxic heavy-metal salts could be replaced by simple buffers such as citrate buffer at pH 6.0 [33]. Heat, rather than microwaves, is important in the retrieval process, since boiling the sections in a pressure cooker [38] or autoclaving them in a buffer solution achieved the same effect. A possible explanation of the process was put forward by Morgan et al.,1994 [35], who suggested that heating not only ruptures the hydroxyl bonds formed by the fixative with the protein antigen, freeing some antigens, but also provides energy to release tissue-bound calcium ions, which contributes to tighter bonds with the fixative. They showed that the salt solutions in which the sections are heated are, in fact, all able to chelate or precipitate calcium to varying degrees, and thus to remove released calcium from the sections, permanently breaking the fixative bonds and revealing antigens. The most effective solutions (EDTA, EGTA) are also the best calcium chelators. The acidity of the buffer may also play a part in antigen retrieval [35,36].

5.2.4.2 Reaction

For the immunohistochemistry procedure, the tissue must be fixed, washed, dehydrated, embedded and sectioned as for conventional histology. Next, the sections, depending on the antibody chosen, must be treated with the specific enzyme(s) for a minimum of 2 hours at 37ºC in a suitable buffer. The enzymes used for GAG reactions are listed in item 7. It is important that the buffer does not evaporate, because the enzymes need the buffer to be efficient. Next, the sections are incubated with the primary antibody in the producer´s recommended dilution for 12- 24 hours. On the next day, the tissue must be incubated with the secondary antibody for 2 hours, washed, and mounted (Figures 1,2).



Figure 1: Diagram of the immunohistochemistry procedure for GAGs.

Figure 2: Immunohistochemical reaction for decorin-specific peptide in the branchial sac of the ascidian Styela plicata. A) Control reaction was obtained omitting the primary antibody; B) Tissue treated with rabbit anti-human decorin-specific peptide antibody. Arrows indicate epithelium cell layer, arrowhead indicates basement membrane and asterisks, the serosa membrane. Scale bars: 50 µm.

6. Immunoelectronmicroscopy

In preparing specimens for transmission electron microscopy, every step of the procedure affects the quality of the final electron micrographs. Therefore, it is important to process tissues according to prescribed methods and to choose the most appropriate method for a particular specimen. Thus, specimen processing should begin with careful planning and according to the nature of the tissue. There is a great variety of methods used in immunoelectronmicroscopy for detecting antigens in biological samples. In general, two main groups of immunochemistry methods are used in transmission electron microscopy: pre-embedding and post-embedding techniques [37]. However, the basic principle is to use an antibody against a target molecule and detect the electron density of tags conjugated to the secondary antibody [22].



6.1 Pre-embedding and post-embedding immunoelectronmicroscopy

Pre-embedding labeling is advantageous for surface labeling of live cells or cell fractions, thick sections of pre-fixed frozen tissue, and vibratome sections. However, this method is limited by the short distance that the antibody can penetrate into the tissue and the large amounts of primary and secondary antibodies that must be used [37,38]. Caution should be taken because the labeling procedure itself may cause redistribution of the antigen on the cell surface. Some fixatives, such as GLA, in low concentrations may stabilize antigens. Because antibodies do not penetrate cell membranes, the cells must be processed with detergents to reveal antigenic sites within the cell. Sometimes, when the tissues are sensitive to fixatives, it is necessary to immunolabel the sample before exposure to fixatives. In this case, certain significant measures should be adopted during the labeling, such as low temperature to avoid patching and capping the antigenic site to be exposed. Then, the fixation can be conducted with a mixture of fixatives and a high concentration of GLA, which gives good structural preservation [37]. In the post-embedding technique, the labeling procedure is carried out directly on tissue sections from embedded specimens. The antigenicity of a sample is partially compromised by the fixation protocol, and the embedding medium itself may influence the antibody binding. The dehydration, infiltration, and embedding steps must be carried out under conditions that optimally preserve antigenicity. In post-embedding procedures, embedding media (LR White, methylacrylate, Lowicryl) must have special characteristics that facilitate the preservation of antigenicity. In addition, small amounts of antibodies are used in this method, compared with pre-embedding procedures [22].

6.2 Fixation

Among the fixatives used for transmission electron microscopy, osmium tetroxide (OsO4) was the first ever employed. Nevertheless, osmium tetroxide is not the best fixative to maintain the ability of the antigen to react with the antibody. There are other fixatives that offer better results for immunoelectronmicroscopy. [22,39].

6.2.1 Marine organisms

6.2.1.1 Fixation

It is important to determine what tissue features are to be evidenced in the study, such as ribosomes, mitochondria, or nuclei. The best results are obtained with 1% GLA in filtered seawater or cacodylate buffer prepared in seawater. In some cases, 0.1 to 0.2% picric acid is used together with PFA and GLA [40]. In general, however, a post-fixation procedure with OsO4 should be avoided because it affects the antigenicity of the sample [41]. The other common difficulty in the fixation of marine organisms is to determine which salts can be used to prepare the seawater solution. If the medium contains significant amounts of reactive salts, such as calcium or magnesium, phosphate buffers will co-precipitate with them, thus potentially inserting precipitates into the tissue, which reduces the buffering capacity of the medium and of the fixative buffer. To avoid this problem, cacodylate buffer or a saline solution without divalent cations (such as Ca+2) are recommended [42]. Under the action of non-coagulant fixatives, such as formaldehyde and GLA, proteins turn into a gel. This is caused by the stabilization of the molecules, which make cross-connections among their components without appreciable distortion of the proteins, and thus of structure. In general, the protocols for chemical fixation use modifications of two basic steps: 1) a primary fixative combining GLA and low concentrations of formaldehyde. This solution allows more rapid initial fixation, because formaldehyde penetrates the tissues better than GLA [28]; 2) a secondary fixative composed of a solution of OsO4 that enhances preservation of membranes and glycogen. In general, however, the secondary fixative is not used in post-embedding procedures because it modifies the final image of the specimen observed under the electron microscope [29]. In recent years, numerous fixative variations based on the GLA, formaldehyde, OsO4 protocol have been developed for particular cells or tissues. We describe here only the fixatives composed of GLA and formaldehyde, because of their capacity to preserve the proteins in the proteoglycans and the disaccharides in GAGs.

6.2.1.1.1 GLA

GLA is the substance used in the majority of studies based on electron microscopy. The aldehyde groups of GLA specifically react with amino groups of lysine in adjacent proteins. In addition, GLA reacts to some degree with lipids, carbohydrates, and nucleic acids [30]. Some carbohydrates, especially glycogen, are retained in the aldehyde-fixed tissue. Approximately 45% to 65% of the total glycogen is retained in tissues after fixation with GLA [31]. However, little information is available about how GAGS are fixed. The rate of penetration of GLA into the tissue is very low. It penetrates more slowly into compact tissue with multiple membrane layers than it does into tissues that have large fluid spaces. A tissue should be fixed in a volume 20 times larger than the sample size [39].



If a buffering system is not used with the primary fixative, the pH of the tissue is drastically lowered during the fixation procedure. As a consequence, numerous artifacts may be produced. Buffering systems that maintain physiological pH result in fewer artifacts. For this reason, the literature should be consulted to obtain the physiological pH for a particular biological system. Cacodylate and phosphate are the main buffers used. Phosphate buffer has been reported to give good results in primary fixations, when aldehyde fixatives are used. Phosphate buffer is often the buffer of choice, since it is nontoxic and physiologically compatible with the cells. Just as important as the selection of the buffer is the osmolarity of the buffering system, since it contributes to the osmolarity of the overall fixative. The total osmolarity of the fixatives includes that of the buffer, that of any added substance (such as NaCl), and that of the fixative [22]. Immunocytochemical reactions traditionally take place in a buffer solution that stabilizes the antibody and does not damage the tissue substrate. The latter condition is more important for fresh than for fixed tissue. The pH must be higher than 7.0 to prevent detachment of antibodies from the tissue, which becomes a danger at a low pH, but it can be raised as high as 9.0 if necessary in order to prevent non-specific binding of reagents [19].

6.2.1.2 Washing

Washing is extremely important because it eliminates any free unreacted fixation molecules that remain within the tissue, and therefore it helps to prevent non-specific results or poor post-embedding or pre-embedding results. After primary fixation, the tissue is usually washed in the same buffer vehicle used in the fixation solution. Aldehydes remaining from the primary fixation will be oxidized by osmium tetroxide in a pre-embedding procedure. The protocols can vary from one to 10 washes for 10 or 15 minutes in the buffer. Unreacted GLA diffuses slowly outward from the tissue, as well as inward, so that a minimum of a few hours of washing with at least three changes of buffer is recommended. Several rinses with at least one overnight wash in buffer will eliminate most of the unreacted GLA and/or PFA. [22].

6.2.1.3 Dehydration

Dehydration is the process of replacing the water in cells with a fluid that acts as a solvent between the aqueous environment of the cell and the hydrophobic embedding media. Most embedding media are only partially miscible or completely immiscible with water. Common dehydration agents are ethanol, acetone or methanol, depending on which is the best solvent for the resin. The general philosophy of the dehydration step is to replace water within the tissue progressively, by using a graded series of dehydration agents. Some researchers dehydrate at 4º C to minimize extraction of cellular materials; however, the low temperature introduces the potential problem of water condensation in the dehydration agent. Usually 50% of the agent is the first solvent used after the fixation procedure, followed by 60%, 70%, 80%, and 95%. It is very important not to reach 100% of the dehydration agent in the post-embedding immunoelectronmicroscopy, because the antibody needs to diffuse in the tissue. In the pre-embedding procedure, the dehydration agent is usually acetone, and it is very important to reach a 100% concentration of this agent. This is a critical issue, because most of the commonly used epoxide resins do not polymerize properly if any trace of water is present [22,42].

6.2.1.4 Infiltration of Resin

Infiltration is the process by which dehydrants or transition fluids are gradually replaced by resin monomers. Water-miscible acrylic embedding media are commercially available, and the most commonly used media have proved to be quite useful in retaining biochemical reactivity within samples. Acrylic media exhibit different characteristics from those of epoxide media, and should be handled according to the specific instructions included with every kit [42]. The infiltration process occurs when the solvent, acetone or other, is mixed with the epoxy or acrylic resin and placed in vials with the tissue. Gradually, the resin-solvent ratio is increased until pure resin is used. The pure-resin specimens are transferred into molds or capsules containing the resin, and are finally placed in an oven, where the resin components polymerize to form a solid. During infiltration by the epoxy resin, the vials of tissue are gently agitated on a turntable that positions the tissue vials at about 30º from the vertical, and slowly rotates the tissue. Tissue vials should be closed so that moisture from the air does not enter them [37]. In general, to start the infiltration, two parts of resin solvent to one part of resin are used for one hour for one day; in sequence, one part of resin solvent to one part of resin; one part of resin solvent to two parts of resin; pure resin; and again pure resin to embed the material within the mold.

6.2.1.5 Embedding

Complete and uniform penetration of tissue specimens by a suitable embedding medium is a prerequisite for satisfactory sectioning. This is accomplished through infiltration and embedding. Embedding essentially involves complete impregnation of the interstices of a tissue specimen with the medium. Embedding must precede sectioning



with a microtome, because tissues are not sufficiently rigid to be cut into thin sections without the additional support of a resin matrix. The post-embedding labeling and the embedding medium itself may influence the antibody binding. Dehydration, infiltration, and embedding steps must be carried out under conditions that optimally preserve antigenicity. For the post-embedding procedure, embedding media (LR white, methylacrylate, Lowicryl) with special characteristics (low-temperature embedding) that facilitate the preservation of antigenicity can be purchased [30].

6.2.1.6 Curing of the embedment and sectioning

The final step in embedding is curing the tissue blocks. In the case of acrylics, curing involves linear polymerization of the mixture. With epoxy resins, a chain of polymerized resin is formed, which cross-links to other chains of resin to form an integrated meshwork that completely permeates the tissue. For most resins, increased temperature accelerates the rate of hardening. Ultraviolet light can be used in combination with catalysts to polymerize resins (for example, Lowicryl resin may be hardened at low temperature using ultraviolet light). A period of 12 hours to 3 days is necessary to harden most embedments. If the capsules can be indented with a thumbnail, then there is a strong possibility that they are not fully hardened [22]. Sectioning is carried out in the same way as for conventional electron microscopy.

6.2.1.7 Reaction

Immunocytochemistry is the identification of a tissue constituent in situ by means of a specific antigen-antibody interaction, in which the antibody has been tagged with a visible label. Cell staining is a powerful method to demonstrate both the presence and subcellular location of a particular molecule of interest [1]. This technique is very tolerant. There are many satisfactory variants of the methods. Most dyes and fluorescent compounds used to reveal the immune complex at the light-microscope level are not appropriate tags for use with the electron microscope, because they are neither electron-dense nor do they produce an electron-dense product. The first tag used was the electron-dense protein ferritin to determine the ultrastructural location. Tags used recently include the avidin-biotin system [43] and the currently very popular colloidal gold technique [44]. The types of tags fall into three major categories, based on the nature of the tag. Some tags are organic molecules that possess structured electron opacity, others are enzymes whose reaction product can be detected after the addition of the substrate, and still others are heavy metals that can be observed directly. The main tag used is colloidal gold (Figure 3). Colloidal suspensions of gold can be easily prepared and readily tagged to immunoglobulins. Of particular note is that these discrete, highly electron-dense particles can be made in sizes from approximately 3 nm in diameter upward [44].

Figure 3: Immunoelectronmicroscopy of heparin in test cells from the ascidian Styela plicata. Cells were treated with mouse anti-human heparin GAG antibody. Scale bars: A) 150 nm; B) 60 nm.

7. Identification of GAGs and Proteoglycans

To identify the CS/DS families it is important to use an antibody with previous treatment of the tissue with chondroitinase ABC (which degrades CS or DS) or chondroitinase AC (which only degrades CS). However, an antibody that requires no enzyme treatment may be chosen. In the former case, specific disaccharides and patterns of sulfation that provide information about the heterogeneity of the molecule may be identified [45]. Chondroitinase ABC, also termed chondroitin ABC-lyase, catalyzes the eliminative cleavage of polysaccharides containing (1→4)β-D-hexosaminyl and (1→3)-β-D-glucoronosyl or (1→3)-α-L-iduronosyl linkages, to disaccharides containing 4-deoxy-β-D-gluc-4-enuronosyl groups. It acts on chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan

A B



sulfate, and on hyaluronate with a slower kinetics. Initial rates of degradation of DS, CS and HA are 40%, 20% and 2%, respectively [45]. Chondroitinase AC, also known as chondroitin AC-lyase, is an eliminase that cleaves CS but not DS. The enzyme acts on either sulfated or non-sulfated polysaccharide chains containing (1→4) linkages between hexosamine and glucuronic-acid residues. The reaction yields oligosaccharide products, mainly disaccharides containing unsaturated uronic acids. This enzyme requires the same conditions for its activity as chondroitinase ABC [45,46]. To identify the HS family, a specific sequence of amino acids along with the “stub” in the GAG molecule must be recognized. In the first case, the immunocytochemistry procedure is the same as that which recognizes protein epitopes. However, the stub needs to be revealed by heparinases first, as described below [47]: Heparinases cleave sulfated glycans containing α-(1→4) glycosidic linkages between the glucosamine and uronic acid residues in the HS or Hep polymers. The cleavage proceeds via an elimination reaction, resulting in an unsaturated oligosaccharide containing uronic acid residues. The three forms of heparinases (I, II, III) have specific substrates, as follows:

a) Heparinase I or heparin lyase I: cleaves heparin and HS (relative activity 3:1) at the linkages between hexosamines and O-sulfated IdoA, yielding mainly disaccharides. The enzyme also cleaves antithrombin III, binding pentasaccharide domains in the heparin molecule.

b) Heparinase II or heparin lyase II: cleaves HS, and to a lesser extent, heparin (relative activity about 2:1), at the (1→4) linkages between hexosamines and uronic-acid residues, both GlcA and IdoA, yielding mainly disaccharides.

c) Heparinase III cleaves at the (1→4) linkages between hexosamine and GlcA residues in HS, yielding mainly disaccharides. The enzyme is not active toward either heparin or heparins of lower molecular weight.

The optimum pH range for the activity of these enzymes is 7.0 to 8.0, the temperature range is 20º to 37º C, and the buffer used is 50 mM sodium phosphate [48,49]. The mammalian hyaluronidases cleave hyaluronic acids and GAGs by hydrolysis. The enzyme from Streptomyces is a lyase that catalyzes cleavage by an elimination reaction, yielding 4-deoxy-4,5-unsaturated oligosaccharides. Its specificity towards other GAGs is unclear. Additionally, there are other types of hyaluronidases that cleave HA and that can be used; however, to identify HA by immunohistochemistry or immunoelectronmicroscopy it is not necessary to use hyaluronidases [45]. Keratanases catalyse the cleavage of β-galactosidic linkages of non-sulfated residues in KS. For bovine KS, this results in a disaccharide monosulfate as the major product. One unit of this enzyme catalyzes the release of 1.0 µmol of a reducing group such as galactose from keratan sulfate per minute at 37ºC, pH 7.4 [50]. The commercial antibodies that are mainly used in research to identify GAGs or proteoglycans are listed below:

a) CS and DS family:

Specificity Immunogen

Ventral membranes of gizzard fibroblast CS

Proteoglycan from chick embryo limb bud Chondroitin-2,6-sulfate

Adult rat bone protein Chondroitin-6-sulfate

Chicken type IX collagen containing chondroitin-4-sulfate Chondroitin-4-sulfate

CSPG digested with chondroitinase ABC ΔDi-0S

Same as above ΔDi-4S

Same as above ΔDi-6S

PG from 10-day-old rat brain Chondroitin-4-sulfate

Extract of monkey brain Chondroitin

In order to identify GAGs in marine organisms, an antibody is generally used, which recognizes a stub sequence which can provide information about sulfation in the CS or DS molecules, and which recognizes ΔDi-0S, Δ Di-4S, and Δ Di-6S as immunogens. In this case, chondroitinase AC can be used to identify the degree of sulfation in CS; or chondroitinase ABC, to identify these degrees in DS.



b) HS family:


Capsular polysaccharides from E. coli K5 HS

HS proteoglycan from rat glomerular basement membrane HS

Mouse fibrocarcinoma induced by Meth-A HS

HSPG from human fetal lung fibroblast HS

HSPG from human fetal lung fibroblast digested with heparinase ΔHS

The antibodies used to identify HS or Hp are immunoreactive only to GlcA-GlcNAc and cannot be used to identify CS, KS or DS. The listed antibodies cannot identify the pattern of sulfation, and other methods must be used to obtain this information. Other non-commercial antibodies were developed to identify Hp from mammal epitopes found in mast cells (ST-1). These antibodies recognize an epitope in the intact unmodified molecule of Hp. No cross-reactivity is generally observed for ST-1 with other GAGs, such as HS, CS, or DS [51].

c) KS family:


CSPG monomer from human articular cartilage digested with chondroitinase ABC KS

A proteoglycan core antigen prepared by chondroitinase ABC digestion of human adult cartilage proteoglycan monomer

KS

To identify KS, chondroitinase ABC and keratanase together or alone can be used. They help to verify the immunoreactivity of the antibodies described in the KS family table, which identify a sequence of amino acids in KS after digestion. In addition, the antibodies against ΔDi-0S, ΔDi-4S and ΔDi-6S, in the CS and DS family table, can be used to verify the degree of sulfation after digestion with these enzymes. The immunoreactivity to the antibodies can be enhanced, depending on the enzyme used (chondroitinase ABC or keratanase), and this reflects the heterogeneity of the KS molecule [52].

d) Core protein of proteoglycan:


Human ovarian fibroma DS proteoglycan (decorin)

Human yolk-sac tumor Large proteoglycan (versican)

HSPG from mouse EHS tumor HS proteoglycan (perlecam)

CSPG from 10-day-old rat brain CS proteoglycan (neurocan)

PG from 10-day-old rat brain CS proteoglycan (phosphacan)

Membrane-bound CSPG purified from 10-day-old rat brains Neuroglycan C

Human serglycin from mast cells (HpPG) Serglycin

The antibodies described in the above table can only distinguish amino acids from proteoglycans. In general, in order to locate these proteoglycans in the tissue sections using these antibodies, it is not necessary to use any of the enzymes described above.

References

[1] Burry RW. Successful Immunocytochemistry in Biomedical Research. 1st ed. New York, NY: Springer-Verlag; 2010. [2] Coons AH, Leduc EH, Connolly JM. Studies on antibody production. I: A method for the histochemical demonstration of specific

antibody and its application to a study of the hyperimmune rabbit. J. Exp. Med. 1955;102:49-60. [3] Li L, Ly M, Linhardt RJ. Proteoglycan sequence. Mol. Biosyst. 2012;8:1613-1625. [4] Varki A, Cummings R, Esko J, Freeze H, Hart G, Marth J. Essentials of Glycobiology. 1st ed. Cold Spring Harbor, NY: Cold

Spring Harbor Laboratory Press; 1999. [5] Volpi N. Anti-inflammatory activity of chondroitin sulfate: new functions from an old natural macromolecule.

Inflammopharmacology. 2011;19:299-306. [6] Rudd TR, Skidmore MA, Guerrini M, Hricovini M, Powell AK, Siligardi G, Yates EA. The conformation and structure of GAGs:

Recent progress and perspectives. Curr. Opin. Struct. Biol. 2010;20:567-674. [7] Zhang L. Glycosaminoglycan (GAG) biosynthesis and GAG-binding proteins. Prog. Mol. Biol. Transl. Sci. 2010;93:1-17.



[8] Saravanan R, Vairamani S, Shanmugan A. Glycosaminoglycans from marine clam Meretrix meretrix (Linne.) are an anticoagulant. Prep. Biochem. Biotechnol. 2010;40:305-315.

[9] Vilela-Silva AC, Werneck CC, Valente AP, Vacquier VD, Mourão PA. Embryos of sea urchin Strongylocentrotus purpuratus synthesize a dermatan sulfate enriched in 4-O and 6-O-disulfated galactosamine units. Glycobiology. 2001;11:433-440.

[10] Gandra M, Cavalcante M, Pavão M. Anticoagulant sulfated glycosaminoglycans in the tissues of the primitive chordate Styela plicata (Tunicata). Glycobiology. 2000;10:1333-1340.

[11] Yamada S, Sugahara K, Özbec S. Evolution of glycosaminoglycans: comparative biochemical study. Commun. Integr. Biol. 2011;4:150-158.

[12] Pavão MS. Structure and anticoagulant properties of sulfated glycosaminoglycans from primitive chordates. Anais Acad. Bras. Ciênc. 2002;74:105–112.

[13] Cooper S, Bennett, W, Andrade J, Benjamin E, Reubinoff J, Martin FP. Biochemical properties of a keratin sulfate/chondroitin sulfate proteoglycan expressed in primate pluripotent stem cell. J. Anat. 2002;200:259-265.

[14] Harlow E, Lane D. Antibodies: A Laboratory Manual. 1st ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1998.

[15] Goding JW. Monoclonal Antibodies: Principles and Practice: Production and Application. 2nd ed. San Diego: Academic Press Limited; 1993.

[16] Guo Z, Boons Z-J. Carbohydrate-Based Vaccines and Immunotherapies. 1st ed. New Jersey: John Wiley & Sons; 2009. [17] Muijen GNP, Van Kuppevelt TH. Human single chain antibodies against heparin: Selection, characterization and effect on

coagulation. Blood. 2011;99:2427-2433. [18] Corley RB. A Guide to Methods in the Biomedical Sciences. 1st ed. New York, NY: Springer; 2005. [19] Javois LC. ed. Immunocytochemical Methods and Protocols. 2nd ed. Methods in Molecular Biology Vol. 115. Totowa, NJ:

Humana Press; 1999. [20] Guesdon JL, Ternynck T, Avrameas S. The uses of avidin-biotin interaction in immunoenzymatic techniques. J. Histochem.

Cytochem. 1979;27:1131-1139. [21] Goldby P. Introduction to Immunocytochemistry. 2nd ed. Polak JM, Van Noorden S. eds. New York, NY: Springer-Verlag;

1997. [22] Bozzola JJ, Russell LD..Electron Microscopy: Principles and Techniques for Biologists. 2nd ed.. Sudbury, MA: Jones and

Bartlett Publishers; 1999. [23] Palay SL, McGee-Russell SM, Gordon Jr. S, Grillo MA. Fixation of neural tissues for Electron microscopy by perfusion with

solutions of osmium tetroxide. J. Cell Biol. 1962;12:385-410. [24] de Barros CM, Andrade LR, Allodi S, Viskov C, Mourier PA, Cavalcante MC, Straus AH, Takahashi HK, Pomin VH, Carvalho

VF, Martins MA, Pavão MS. The hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma. J. Biol. Chem. 2007;282:1615-1626.

[25] Gomes AM, Kozlowski EO, Pomin VH, de Barros CM, Zaganeli JL, Pavão MS. Unique extracellular matrix heparan sulfate from the bivalve Nodipecten nodosus (Linnaeus, 1758) safely inhibits arterial thrombosis after photochemically induced endothelial lesion. J. Biol. Chem. 2010;285:7312-7323.

[26] Gilkey JC, Staehelin A. Advances in ultrarapid freezing for the preservation of cellular ultrastructure. J. Electr. Microsc. Tech. 1986;3:177-210.

[27] Sesso A. Fixação de Sistemas Biológicos. In: Técnicas Básicas de Microscopia Eletrônica Aplicadas às Ciências Biológicas. 1st ed. Souza, W. ed. Rio de Janeiro: Sociedade Brasileira de Microscopia; 1998.

[28] Bishop AE, Polak JM, Bloom SR, Pearse AGE. A new universal technique for the immunocytochemical localization of peptidergic innervation. J. Endocrinol. 1978;77:25-26.

[29] Cima F. Microscopy methods for morpho-functional characterisation of marine invertebrate haemocytes. In: Méndez-Vilas A, Días J, eds. Microscopy: Science, Technology, Applications and Education. Badajoz: Formatex Research Center; 2010:1100-1107.

[30] Hayat MA. Principles and Techniques of Electron Microscopy: Biological Applications. 4th ed. New York, NY: Cambridge University Press; 2000.

[31] Glauert AM. Fixation, dehydration and embedding of biological specimens. In: Practical Methods in Electron Microscopy. Vol. 3, Part 1. Amsterdam: North-Holland; 1975.

[32] Polka JM, Van Noorden S. Introduction to Immunocytochemistry. 2nd ed. New York, NY: Springer Verlag; 1997. [33] Cattoretti G, Suurjmeijer AJH. Antigen unmasking on formalin-fixed paraffin embedded tissues using microwaves: a review.

Adv. Anat. Pathol. 1995;2:2-9. [34] Shi S-R, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for

immunocytochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 1991;39:741-748. [35] Morgan JM, Navabi H, Schmid KW, Jasadi B. Possible role of tissue-bound calcium ions in citrate-mediated high-temperature

antigen retrieval. J. Pathol. 1994;174:301-307. [36] Shi S-R, Imam A, Young L, Cote RJ, Taylor CR. Antigen retrieval immunohistochemistry under the influence of pH using

monoclonal antibodies. J. Histochem. Cytochem. 1993;43:193-201. [37] Padron ST. Imunocitoquímica: Técnicas pré e pós inclusão. In: Técnicas Básicas de Microscopia Eletrônica Aplicadas às

Ciências Biológicas. 1st ed. de Souza W, ed. Rio de Janeiro, RJ: Sociedade Brasileira de Microscopia; 1998. [38] Priestley JV, Alvarez FJ, Averill S. Pre-embedding electron microscopic immunocytochemistry. In: Electron Microscopic

Immunocytochemistry: Principles and Practice. 2nd ed. Polak JM, Priestley JV, eds. Oxford, UK: Oxford University; 1992:89-121.

[39] Palade GE. A study of fixation for electron microscopy. J. Exp. Med. 1952;95:285-298. [40] Florim da Silva, S, Corrêa CL, Tortelote GG, Einicker-Lamas M, Martinez AM, Allodi S. Glial fibrillary acidic protein (GFAP)-

like immunoreactivity in the visual system of the crab Ucides cordatus (Crustacea, Decapoda). Biol. Cell. 2004;96:727-734.



[41] Roth J, Bendayan M, Carlemalm E, Villiger W, Garavito RM. Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J. Histochem. Cytochem. 1981;29:663-671.

[42] Dykstra MJ, Reuss LE. Biological Electron Microscopy, Theory, Techniques and Troubleshooting. 2nd ed. New York, NY: Springer; 2003.

[43] Heitzman H, Richards FM. Use of the avidin-biotin complex for specific staining of biological membranes in electron microscopy. Proc. Natl. Acad. Sci. USA. 1974;71:3537-3539.

[44] Faulk WP, Taylor GM. An immunogold method for the electron microscope: Immunochemistry. 1971;8:1081-1083. [45] Ernst S, Langer R, Cooney CL. Enzymatic degradation of glycosaminoglycans. Critical Reviews in Biochemistry and Molecular

Biology. 1995;30:387-444. [46] Gu K, Linhard, RJ, Laliberté M, Gu K, Zimmermann J. Purification, characterization and specificity of chondroitin lyases and

glycuronidase from Flavobacterium heparinum. Biochem. J. 1995;312:569-577. [47] McLean MW, et al. Purification by further chromatography. Eur. J. Biochem. 1984;145:607-615. [48] Linhardt RJ, et al. Mode of action of heparin lyase on heparin. Biochem. Biophys. Acta. 1982;702:197-203. [49] Yang VC, et al. Purification and characterization of heparinase from Flavobacterium heparinum. J. Biol. Chem. 1985;260:1849-

1857. [50] Horton DS, Michelacci YM. Mucopolysaccharidases from Pseudomonas sp. Isolation and partial characterization of constitutive

enzymes involved in the degradation of keratan sulfate and chondroitin sulfate. Eur. J. Biochem. 1986;161:139-147. [51] Straus, AH, Travassos LR, Takahashi HK. A monoclonal antibody (ST-l) directed to the native heparin chain. Anal. Biochem.

1992:20:l-8. [52] Caterson B, Christner JE, Baker JR. Identification of a monoclonal antibody that specifically recognizes corneal and skeletal

keratan sulfate. J. Biol. Chem. 1983;258:8884-8854.



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