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Mechanisms of Heat-induced Antigen Retrieval : Does pH or Ionic Strength of the Solution Play a Role for Refolding Antigens?

Katsura Emoto, Shuji Yamashita and Yasunori Okada

Department of Pathology (KE,YO) and Electron Microscope Laboratory (SY), School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan

Correspondence to: Shuji Yamashita, Electron Microscope Laboratory, School of Medicine, Keio University, 35-Shinanomachi, Shinjuku-ku, Tokyo 160-8582. E-mail: shuji{at}sc.itc.keio.ac.jp

	Summary

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We investigated the effects of pH and ionic strength of solutions used for antigen retrieval to elucidate the mechanism of heat-induced antigen retrieval (HIAR) in immunohistochemistry. The immunostaining intensity of nuclear, cytoplasmic, cell membrane, and extracellular matrix antigens with 17 different antibodies was evaluated in formaldehyde-fixed and paraffin-embedded mouse and human tissues. Deparaffinized sections were autoclaved for 10 min in buffers with different pH values ranging from 3.0 to 10.5. To test the influence of ionic strength on immunoreactions, the sections were autoclaved for 10 min in 20 mM Tris-HCl buffers (TB) at pH 9.0 and 10.5 with or without 25, 50, and 100 mM NaCl. There were two immunostaining patterns for pH dependency of HIAR. First, the majority of antibodies recovered their antigenicity when heated in the buffers with both acidic pH (pH 3.0) and basic pH (pH 9.0 and 10.5). Second, some antibodies showed strong immunostaining only at basic pH values (pH 9.0 and 10.5). When the sections were autoclaved in TB at pH 9.0, immunostaining of all eight antibodies examined decreased as the NaCl concentration increased. On the other hand, when the sections were treated with TB at pH 10.5, all antibodies yielded stronger reactions in the buffer containing NaCl than in the buffer without NaCl; five antibodies exhibited the strongest immunoreaction at concentrations from 25 to 50 mM. These results suggest that the extended polypeptides by heating are charged negatively or positively at basic or acidic pH, and that an electrostatic repulsion force acts to prevent random entangling of polypeptides caused by hydrophobic attractive force and to expose antigenic determinants, during cooling process of HIAR solution. (J Histochem Cytochem 53:1311–1321, 2005)

Key Words: heat-induced antigen retrieval • pH dependency • ionic strength dependency • epitope exposure • immunohistochemistry

	Introduction

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ANTIGENICITY OF SOME ANTIGENS is restored after heat treatment, as reported by Shi et al. (1991). Since then, heat-induced antigen retrieval (HIAR) became a common technique in pathology and histology (Cattoretti et al. 1993; Morgan et al. 1994; Werner et al. 1996; Pileri et al. 1997). HIAR was applied to immunohistochemistry of formalin-fixed and paraffin-embedded tissues, but recent studies showed that it is also applicable to frozen sections (Yamashita and Okada 2005b) and ultrathin sections prepared for immunoelectron microscopy (Hann et al. 2001; Brorson 2002; Goode et al. 2004).

Investigators have tried to select the most suitable conditions (temperature, pH, buffers, and additives) for heating each antigen, because the mechanisms of HIAR are not well understood. Based on studies in which purified proteins treated with formaldehyde were analyzed by SDS-PAGE after heating, Rait et al. (2004) and we (Yamashita and Okada 2005a) have recently demonstrated that heat treatment cleaves the intra- and intermolecular crosslinks (i.e., methylene bridges) induced with formaldehyde. We have also demonstrated by Western blotting that ß-actin and fibronectin can be extracted from autoclaved paraffin sections (Yamashita and Okada 2005a). These studies suggest that the main mechanism of HIAR is disruption of methylene bridges.

Citrate buffer (pH 6.0) is the most popular solution for HIAR (Shi et al. 1993; Brown and Chirala, 1995), but several investigators have reported that the efficiency of HIAR is highly dependent on the pH of the retrieval solutions (Shi et al. 1995; Evers and Uylings 1997; Ferrier et al. 1998; Brorson 2002). Shi et al. (1995) reported three patterns of pH-influenced HIAR immunostaining: type A, almost constant staining, with only a slight decrease in staining intensity between pH 3.0 and pH 6.0; type B, a dramatic decrease in staining intensity between pH 3.0 and pH 6.0; and type C, increasing intensity of immunostaining correlated with increasing pH values of the HIAR solution. Yamashita and Okada (2005a) showed that intensity of immunoreactions obtained by heating in buffer is reversibly changed by successive heating in another buffer with different pH. Nine of 10 antigens exhibited much stronger immunoreaction in sections heated in Tris-HCl buffer (TB) at pH 9.0 than in those heated in TB at pH 6.0 (Yamashita and Okada 2005a). A second heating at pH 6.0 significantly decreased immunostaining of the antigens that had been boiled at pH 9.0, but the immunostaining was restored after a third heating at pH 9.0. These findings suggest that the pH of the buffers is a factor that is essential for proper refolding of antigens or exposure of antigenic determinants so that they are capable of reacting with antibodies.

In the present study, we investigated the effect of the pH and ionic strength of the retrieval solution on HIAR in formaldehyde-fixed and paraffin-embedded specimens to elucidate the mechanisms of HIAR in detail.

	Materials and Methods

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Reagents and Tissue Preparation
Table 1 shows the antibodies used in this study and their sources, abbreviations, and dilutions. Tissues from mature CD-1 mice (8 weeks old) were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 6 hr or with 10% formalin for 24 hr, and washed with PBS (10 mM phosphate buffer, pH 7.4 containing 0.85% NaCl) overnight at 4C. They were then dehydrated with graded alcohols, cleared in xylene, and embedded in paraffin. Routinely formalin-fixed (20% formalin), paraffin-embedded sections of human colon and uterus were obtained from file at Department of Pathology, School of Medicine, Keio University.

Immunohistochemical Staining
The sections were treated with PBS containing 1% BSA and 10% block ace (Dainippon Pharmaceutical Ltd.; Osaka, Japan) at room temperature for 1 hr, and then with the primary antibodies overnight at 4C. After washing with PBS, the sections were incubated with labeled polymer horseradish peroxidase anti-rabbit or labeled polymer horseradish peroxidase anti-mouse (DakoCytomation; Carpinteria, CA) for 1 hr at room temperature. Peroxidase enzyme activity was visualized with imidazole-3, 3'-diaminobenzidine tetrahydrochloride solution. Normal rabbit IgG or mouse IgG was used for the controls in place of the primary antibodies. For the control of estrogen receptor (ER), ERß, glucocorticoid receptor (GR), and androgen receptor (AnR) immunostaining, antibodies absorbed with the respective antigenic peptides were used. Some sections were counterstained with hematoxylin.

Influence of Ion Strength in Immunohistochemistry
Tissues fixed with 4% paraformaldehyde for 6 hr were used in this examination. Deparaffinized sections were autoclaved for 10 min in 20 mM TB (pH 9.0 and pH 10.5) containing 0 mM, 25 mM, 50 mM, or 100 mM NaCl, and immunohistochemical staining was carried out according to the procedures described previously. Staining for eight antigens located in the nucleus, cytoplasm, cell membrane, or extracellular matrix was assessed in this study: ER, AnR, GR, ß-actin, tubulin, claudin-5, fibronectin, and laminin.

	Results

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Effects of pH on Cell Structures
When sections were autoclaved in glycine-HCl buffer, pH 3.0, the nuclear components of several cell types were extracted from the tissues fixed with 4% paraformaldehyde for 6 hr; in particular, the nuclei of the epithelial cells of the genital tract and intestine and of the liver cells were severely damaged (Figure 1B): in the tissues fixed with formalin for 24 hr, partial destruction of nuclear structure was observed in these cells. Small amounts of secreted materials remained in the lumens or ducts of the coagulating gland, seminal vesicles, and pancreas, and the cytoplasmic staining with eosin in many cell types was weak in the autoclaved sections at pH 3.0 compared with unautoclaved sections (Figures 1A and 1B). The nuclear components of some cell types were partially extracted in the specimens fixed for 6 hr after autoclaving in TB at pH 10.5 (Figure 1F). In the human specimens, the nuclear structure was preserved after autoclaving in any buffers. As the pH of the buffers was more basic, staining with hematoxylin became weaker than in sections treated with buffers at pH 4.5, pH 6.0, and pH 7.5 (Figures 1B–1F).

View larger version (155K): [in this window] [in a new window]   Figure 1 Effects of pH on cell structures. Mouse epididymis was fixed with 4% paraformaldehyde for 6 hr and embedded in paraffin. Deparaffinized sections were autoclaved at 120C for 10 min in the following buffers: 20 mM glycine-HCl buffer, pH 3.0 (B), 20 mM citrate buffer, pH 4.5 (C) and pH 6.0 (D), and 20 mM Tris-HCl buffer (TB) at pH 9.0 (E) and pH 10.5 (F). Allowing them to cool to room temperature, the sections were washed with PBS and then stained with hematoxylin and eosin. The section without autoclaving was shown as a control (A). When the section was autoclaved at pH 3.0, the nuclei of epithelial cells are severely destroyed (B). In the section autoclaved at pH 10.5, nuclear components of some cells are extracted (F). As pH of buffers becomes more basic, staining with hematoxylin becomes weaker (B–F). Bar = 25 µm.

View larger version (121K): [in this window] [in a new window]   Figure 2 Effects of pH on heat-induced antigen retrieval. Sections from tissues fixed with 4% paraformaldehyde for 6 hr were autoclaved at 120C for 10 min in the following buffers and then immunostained: 20 mM glycine-HCl buffer (pH 3.0) (B,G,L,Q), 20 mM citrate buffer (pH 6.0) (C,H,M,R), 20 mM TB at pH 9.0 (D,I,N,S), and pH 10.5 (E,J,O,T). Sections without autoclaving were used as control sections (A,F,K,P). Estrogen receptor ß (ERß) (A–E), progesterone receptor (PR) (F–J), laminin (K–O), and lysozyme (P–T) were localized in the mouse ovary (A–E), uterus (F–J), kidney (K–O), and small intestine (P–T), respectively. Sections immunostained with antibody to laminin (K–O) were counterstained with hematoxylin. ERß exhibits a strong immunostaining in the nuclei of granulosa cells when autoclaved at pH 9.0. (D) Weak reaction is seen when heated at pH 3.0 (B). PR shows positive immunoreaction in the stromal cells at pH 3.0 (G), and in the glandular epithelia and stromal cells at pH 9.0 (I). Immunoreaction for laminin is seen along the basal lamina when autoclaved at pH 3.0 (L), 9.0 (N), or 10.5 (O). Lysozyme shows positive immunoreaction in the Paneth cells in the sections autoclaved at pH 9.0 (S) and pH 10.5 (T), but negative reaction in the sections treated at pH 3.0 (Q) and pH 6.0 (R). Bars: A–E = 25 µm; F–T = 50 µm.

View larger version (128K): [in this window] [in a new window]   Figure 3 Effects of pH on heat-induced antigen retrieval in the human tissues. Progesterone receptor (PR) (A–G) and E-cadherin (H–N) were localized in the human uterus (A–G) and colon (H–N) routinely fixed with 20% formalin and embedded in paraffin, respectively. Sections were autoclaved at 120C for 10 min in the following buffers and then immunostained: 20 mM glycine-HCl buffer (pH 3.0) (B,I), 20 mM citrate buffer (pH 4.5) (C,J), 20 mM citrate buffer (pH 6.0) (D,K), 20 mM TB at pH 7.5 (E,L), pH 9.0 (F,M), and pH 10.5 (G,N). Sections were immunostained without heating (A,H). Strong PR immunostaining is present in the nucleus of glandular epithelium and stromal cells when the sections were autoclaved at pH 3.0 or pH 9.0. E-cadherin is localized along the cell membrane of intestinal glandular epithelium in the colon autoclaved at pH 9.0 and pH 10.5. Bar = 50 µm.

View larger version (138K): [in this window] [in a new window]   Figure 4 Effects of ionic strength on heat-induced antigen retrieval. Claudin-5 (A–H) and glucocorticoid receptor (GR) (I–P) were immunolocalized in the mouse kidney and the liver, respectively. The sections were autoclaved at 120C for 10 min in 20 mM TB at pH 9.0 with 25 mM NaCl (B,J), 50 mM NaCl (C,K), or 100 mM NaCl (D,L) or without NaCl (A,I), and in TB at pH 10.5 with 25 mM NaCl (F,N), 50 mM NaCl (G,O), or 100 mM NaCl (H,P) or without NaCl (E,M). Immunostaining of claudin-5 in the endothelial cells of glomerulus and blood vessel and of GR in the nuclei of liver cells is the strongest in the section autoclaved in NaCl-free TB at pH 9.0 (A,I), and in the sections autoclaved in TB containing 50 mM NaCl at pH 10.5 (G,O). Bar = 50 µm.

View larger version (30K): [in this window] [in a new window]   Figure 5 Profile of effects of ionic strength to heat-induced antigen retrieval. Sections were treated according to the procedure described in Figure 4. The following antigens were immunostained: estrogen receptor in the uterus; androgen receptor in the epididymis; tubulin in the small intestine; claudin-5 in the kidney; glucocorticoid receptor in the liver; ß-actin in the small intestine; laminin in the kidney; and fibronectin in the liver. Assessment of immunostaining with a scale (0–3) was used: 0, negative; 1, weak; 2, moderate; 3, strong. Dotted and solid lines display immunostaining intensity of antigens autoclaved in TB (pH 9.0) and TB (pH 10.5) containing various concentrations of NaCl, respectively. All antigens show the strongest immunoreaction when sections were autoclaved in NaCl-free TB at pH 9.0 (dotted lines), but they yield the strongest staining when sections were heated in TB containing NaCl at pH 10.5 (solid lines).

	Discussion

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Many biochemical studies have demonstrated that unfolded polypeptides treated with denaturants, such as urea or guanidine hydrochloride, readily self-associate or randomly associate with other proteins in the solution on removal of the denaturants. Hydrophobic force is thought to be the principal driving force for the protein aggregation (Jaenicke and Seckler 1997; Fink 1998; Hanson and Gellman 1998). Polypeptides are tightly packed and neighboring polypeptides crossly contact each other in the tissues, particularly in the nuclei, secretory granules, and fibrous structures. We hypothesized that the pH dependency of HIAR is based on the following mechanisms (Figure 6). When methylene bridges are cleaved by heating, the higher order structure of antigen molecules is destroyed and polypeptide chains extend to expose both hydrophobic and hydrophilic portions. Antigenic determinants are concealed at around neutral pH, because attractive forces caused by the hydrophobic and ionic forces act strongly and entangle neighboring polypeptides randomly as the solutions cool. At basic or acidic pH values, however, the majority of extended polypeptides are charged negatively or positively and an electrostatic repulsion force acts to prevent random aggregation of polypeptides based on the hydrophobic force.

View larger version (39K): [in this window] [in a new window]   Figure 6 Schematic of heat-induced antigen retrieval mechanisms. After heating, methylene bridges produced by formaldehyde are cleaved and polypeptide chains extended to expose their hydrophobic regions. During cooling process, the polypeptide chains rapidly refold. At pH 3.0 and pH 9.0, hydrophobic attractive force and electrostatic repulsion force based on positively or negatively charged polypeptides may balance to prevent intertwining of polypeptide chains and expose antigenic determinants for antigen–antibody interaction. On the other hand, at pH 4.5–7.5, ionic and hydrophobic attractive forces may cooperatively act and neighboring polypeptides entangle each other to hidden antigenic determinants.

To test the hypothesis described previously, we immunostained sections heated in TB at pH 9.0 or pH 10.5 containing 25–100 mM NaCl to reduce the electrostatic forces. At pH 9.0, all antigens exhibited the strongest immunostaining when autoclaved in TB without NaCl. At pH 10.5, on the other hand, all antigens showed stronger immunostaining when heated in TB containing NaCl than in NaCl-free TB. Five antigens exhibited the strongest immunostaining when heated in TB containing 25–50 mM NaCl. Stempfer et al. (1996) reported similar results using in vitro system in which -glucosidase with extended hexa-arginine peptide was denatured with 8 M urea and refolded on a polyanionic matrix. Refolding was only feasible at moderate salt concentrations (30–40 mM NaCl) at pH 7.6. Refolding of the enzyme was prevented by strong ionic attraction between the protein and the matrix, when the denaturant was removed from the solution at very low salt concentration. At high salt concentrations (>100 mM NaCl), refolding was inhibited by hydrophobic interactions between unfolded or partially folded protein and the matrix. The refolding process of polypeptides in the tissues may more closely resemble the system with the matrices rather than in solution.

These biochemical findings support our hypothesis concerning pH-dependent HIAR. At around pH 9.0, there may be a balance between the hydrophobic force and the ionic repulsion force by negatively charged proteins that prevents intertwining of unfolded polypeptide chains and maintains a suitable conformation of many epitopes to interact with antibodies in the tissues. At pH 9.0, increasing ionic strength reduces electrostatic repulsion and the hydrophobic force causes the polypeptide to become entangled and subsequently reduces immunostaining. At pH 10.5, on the other hand, reduction of electrostatic force in the buffer containing NaCl probably moderates the denaturation of polypeptides based on the highly negatively charged polypeptide chains and yields stronger immunoreactions than in buffer that does not contain NaCl. Recently, Namimatsu et al. (2005) have demonstrated that heating in citraconic anhydride solution at neutral pH is a universal antigen retrieval method. Their results may also support our hypothesis. Citraconic anhydride modifies -amino groups of lysine residues, places numerous negative charges on proteins at neutral pH, and elicits conformational changes of proteins (Dixon and Perham 1968; Batra 1991; Mir et al. 1992). In their antigen-retrieval method, it is postulated that heating exposes -amino groups of lysine modified with formaldehyde and the -amino groups are citraconylated, and that the proteins in the tissues are charged negatively at neutral pH. Then, the citraconyl groups may be gradually removed from proteins. Therefore, electrostatic repulsion by negative charges and hydrophobic attraction may have a balance to expose epitopes during citraconylation at neutral pH.

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid (16590154) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

We are grateful to Satoshi Kusakari, Hitoshi Abe, Minako Suzuki, Kiyora Nakajima, and Yuko Hashimoto for their technical assistance.

	Footnotes

 
Received for publication January 21, 2005; accepted June 23, 2005

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 

Batra PP (1991) Regeneration of native ovalbumin conformation disrupted by citraconylation. Biochimie 73:1397–1402[Medline]

Brorson SH (2002) pH-dependent effect of heat-induced antigen retrieval of epoxy section for electron microscopy. Micron 33:481–482

Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol 8:515–520[Medline]

Cattoretti G, Pileri S, Parravicini C, Becker MH, Poggi S, Bifulco C, Key G, et al. (1993) Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol 171:83–98[CrossRef][Medline]

Dixon HBF, Perham RN (1968) Reversible blocking of amino groups with citraconic anhydride. J Biochem 109:312–314

Evers P, Uylings HB (1997) An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative. J Neurosci Methods 7:197–207

Ferrier CM, van Geloof WL, de Witte HH, Kramer MD, Ruiter DJ, van Muijen GN (1998) Epitopes of components of the plasminogen activation system are re-exposed in formalin-fixed paraffin sections by different retrieval techniques. J Histochem Cytochem 4:469–476

Fink AL (1998) Protein aggregation: folding aggregates, inclusion bodies and amyloid. Fold Des 3:R9–23[CrossRef][Medline]

Goode NP, Shires M, Crellin DM, Khan TN, Mooney AF (2004) Post-embedding double-labeling of antigen-retrieved ultrathin sections using a silver enhancement-controlled sequential immunogold (SECSI) technique. J Histochem Cytochem 52:141–144[Abstract/Free Full Text]

Hann CR, Springett MJ, Johnson DH (2001) Antigen retrieval of basement membrane proteins from archival eye tissues. J Histochem Cytochem 49:475–482[Abstract/Free Full Text]

Hanson PE, Gellman SH (1998) Mechanistic comparison of artificial-chaperone-assisted and unassisted refolding of urea-denatured carbonic anhydrase B. Fold 3:457–468

Jaenicke R, Seckler R (1997) Protein misassembly in vitro. Adv Protein Chem 50:1–59[Medline]

Mir MM, Fazili KM, Qasim MA (1992) Chemical modification of buried lysine residues of bovine serum albumin and its influence on protein conformation and bilirubin binding. Biochim Biophys Acta 1119:261–267[CrossRef][Medline]

Morgan JM, Navabi H, Schmid KW, Jasani B (1994) Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol 174:301–307[CrossRef][Medline]

Namimatsu S, Ghazizadeh M, Sugisaki Y (2005) Reversing the effect of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method. J Histochem Cytochem 53:3–11[Abstract/Free Full Text]

Pileri SA, Roncador G, Ceccarelli C, Piccioli M, Briskomatis A, Sabattini E, Ascani S, et al. (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183:116–123[CrossRef][Medline]

Rait VK, Xu L, O'Leary TJ, Mason JT (2004) Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment. Lab Invest 84:300–306[CrossRef][Medline]

Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem 41:1599–1604[Abstract]

Shi SR, Cote RJ, Young L, Imam A, Taylor CR (1996) Use of pH 9.5 Tris-HCl buffer containing 5% urea for antigen retrieval immunohistochemistry. Biotech Histochem 72:190–196

Shi SR, Imam SA, Young L, Cote RJ, Taylor CR (1995) Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 43:193–201[Abstract]

Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741–748[Abstract]

Stempfer G, Holl-Neugebauer B, Rudolph R (1996) Improved refolding of an immobilized fusion protein. Nat Biotechnol 14:329–334[CrossRef][Medline]

Werner M, Von Wasielewski R, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105:253–260[CrossRef][Medline]

Yamashita S (1981) Immunohistochemical study of amylase and deoxyribonuclease in rat parotid gland. Acta Histichem Cytochem 14:236–260

Yamashita S, Okada Y (2005a) Mechanisms of heat-induced antigen retrieval: a study in vitro employing SDS-PAGE. J Histochem Cytochem 53:13–21[Abstract/Free Full Text]

Yamashita S, Okada Y (2005b) Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections. J Histochem Cytochem (Epub ahead of print)

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This Article Abstract Full Text (PDF) All Versions of this Article: jhc.5C6627.2005v1 53/11/1311    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Emoto, K. Articles by Okada, Y. Search for Related Content PubMed PubMed Citation Articles by Emoto, K. Articles by Okada, Y. Social Bookmarking                      What's this?

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