新しい光顕免疫染色法(Protein A gold-silver染色法)の開発に関する研究 Application of Protein A gold for light microscopic immunohistochemistry in paraffin sections : Protein A gold-silver staining method

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著者

    • 藤森, 修 フジモリ, オサム

書誌事項

タイトル

新しい光顕免疫染色法(Protein A gold-silver染色法)の開発に関する研究

タイトル別名

Application of Protein A gold for light microscopic immunohistochemistry in paraffin sections : Protein A gold-silver staining method

著者名

藤森, 修

著者別名

フジモリ, オサム

学位授与大学

麻布大学

取得学位

獣医学博士

学位授与番号

乙第263号

学位授与年月日

1987-12-02

注記・抄録

博士論文

プロテインAゴールド(PAG)法は主として電顕免疫組織化学の染色洪として用いられている方法であり,種々の動物のIgG-Fcフラグメントと結合する能力をもつプロテインAの性質を利用し,抗原-一次抗体反応部位において一次抗体のFcフラグメントにプロテインAを結合させ,これに標識されているコロイド金粒子を電子顕微鏡下で観察して抗原の局在を間接的に検索するものである。PAG法は電顕的免疫組織化学の有効な染色技術として多用されているが,これを光顕免疫組織化学の染色法として応用する試みもなされ,Roth(1982)によって開発された光顕的PAG染色法は一応の成果を上げてはいるが,用いる一次抗体およびPAGの濃度が高く,反応産物の色調も淡いため広く利用され得る方法ではなかった。この度著者はPAG法を光顕免疫組織化学染色の有効な方法として活用するために,PAG法に物理現像法を併用し従来の光顕的PAG染色法の欠点を改善した新しい染色法の開発を試みた。なお染色法の開発に当たっては,従来の物理果像液にみられる非特異的な金属銀の析出等の問題点を改善した物理現像液を考案した。 組織材料にはブアン液にて固定したラット,イヌ,サルおよびヒトの膵尾部の組織をパラフィン切片として用い,一次抗体には抗インスリン・モルモット血清及び抗グルカゴン,抗ソマトスタチン,抗膵ペプチドのウサギ血清,計4種を使用,PAGは金粒子の径が10nmおよび5nmの2種類を用いた。本染色法に使用する改良物理現像液の組成は以下の通りである。A液:20%精製アラビアゴム水溶液,45ml。10%硝酸銀水溶液,1ml。B液:ブロモハイドロキノン,200mg。クエン酸,300mg。蒸留水,15ml。現像液は使用時にA,B両液を混合して作成し,現像は20℃の暗黒中で行なった。対照染色としては吸収試験および阻止試験を行なって染色の特異性を検討し,さらに現像液の特性を比較するために他の物理現像液を用いた染色も試みた。 ラットの膵臓で,抗インスリン抗体と金粒子の径10nmのPAGを用いて行なったProtein A gold-silver(PAGS)染色の結果によれば,膵島中心性に黒色の強い反応を示す細胞集団が認められ,対照染色の結果からこれらの反応産物はインスリン分泌細胞に特異的に反応していることが明かであった。即ち物理現像を施すことにより抗原局在部位に結合する金粒子に金属銀を沈着させ,これを黒色の反応産物として光顕的に可視化することが可能になった。その染色所見では反応産物と組織とのコントラストは極めて鮮明であり,本染色法が反応の増強性と反応産物の可視性に優れた方法であることを示唆していたが,反応の強度はむしろ強すぎる傾向にあり,使用した一次抗体及びPAGの濃度も比較的高濃度であるため,適度な染色強度と高い感度を有する染色法とするためにさらに改良を行なった。また一般的な免疫染色法として活用されるためには幅広い適用性が要求されるので,このことについて検討するために他の抗体を用いる免疫染色および他の動物組織を用いる免疫染色,さらに酵素抗体法との組み合わせによる二重染色も行なった。その結果,10nmのPAGを用いる染色ではインスリン抗体濃度:2000倍,PAG濃度:80倍で70~80分目現像により適度な反応強度が得られ,かつ非特異反応の極めて少ないPAGS染色が可能であった。さらに本染色法の感度を向上させるために金粒子の径が5nmのPAGを用いて染色を試みたところ,本染色法の感度は飛躍的に向上した。また他の膵島ホルモン抗体を用いてラットの膵島で本染色法による免疫染色を試みたところ,いずれの抗体を用いた染色においてもインスリン抗体による染色と同様の優れた染色結果を得ることができた。さらに各膵島ホルモン抗体を用いてイヌ,サルおよびヒトの膵島について染色を試みても同様の結果を得ることができた。現在免疫染色法として最も一般的な間接酵素抗体法などのimmunoperoxidase法と比較すると,本染色法では用いる一次抗体および二次抗体に相当するPAGの希釈倍率では同等あるいは数倍の感度を有し,またコントラストの高い黒色顆粒状の強い反応産物はその可視性の点においてむしろ優れていた。さらにPAGS染色法と酵素抗体法を組み合わせてA細胞とB細胞の二重染色を試みたところ,A細胞はPAGS染色法によって黒色に,B細胞は酵素抗体法によって茶褐色に染め分けられ,両染色法による反応産物は色調の対比が明瞭で,相互の識別が容易であった。その上,α-ナフトールとDABを発色剤として使用する酵素抗体法による従来の二重染色法とは異なり,本二重染色法では通常の脱水,封入が可能であり標本の保存性が高かった。 なお対照染色では,各抗体を対応する抗原で吸収した吸収試験とPAGの一次抗体への結合をブロックした阻止試験はともに反応が陰性であり,本染色法の有する高い特異性が確認された。 改良物理現像液にかえて,物理現像液として代表的なDanscherの現像液およびその一変法であるMoeremansの現像液をPAGS染色法に適用して現像液の比較を行なったところ,2種の現像液のいずれを用いても本染色法特有の染色結果は得られず,改良現像液は現像力の強さと現像力の持続性ならびに非特異反応が少ないなどの点で他の物理現像液に比べ優れていることが裏付けられた。従って本染色法の特徴である反応の強い増強性と高い特異性は改良物理現像液の特性に起因するものである。本現像液では還元剤としてブロモハイドロキノンを使用し,保護コロイドのアラビアゴムを超遠心によって精製して用いている。ブロモハイドロキノンはハイドロキノンに比較して極めて強い還元力を有しているため本現像液の現像力は著しく高まり,かつ精製アラビアゴムを用いることで現像液の自己触媒作用が抑えられて非特異的な金属銀の析出を長時間にわたって防止できた。これらのことが,極めて微量な金粒子を光顕的に可視化するために必要な,強い現像力と長時間の現像を可能にしたものと考えられた。 本研究において確立された10nmまたは5nmのPAGSを用いるPAGS染色法の基本的染色要領は下記に示す通りである。 1. 切片を脱パラフィンする。 2. 0.01M燐酸緩衝食塩液(PBS)にて5分間3回,計15分洗浄する。 3. 5%卵白アルブミンPBS溶液にて湿箱中で処理する。30分。 4. 一次抗体と湿箱中で60~90分反応させる。 5. PBSで抗体を洗い流し,さらにPBS中で10分3回,計30分洗浄する。 6. 5%卵白アルブミンPBS容液にて湿箱中て15分処理する。 7. PAG液と湿箱中で60分反応させる。 8. 0.01M燐酸緩衝液(PB)にてPAGを洗い流し,さらにPB中で10分3回,計30分洗浄する。 9. 暗黒中て20℃にて物理現像を行なう。現像の結果の判定を行なうには,現像液より切片を取り出し流水で1分水洗し検鏡して行なう。現像が不充分の場合は蒸留水で洗った後,再度現像液に戻して現像を行なう。 10. 流水中で5分水洗する。 11. 5倍に希釈した写真用定着液にて1分定着する。 12. 流水中で10分水洗する。 13. ケルンエヒトロートで軽く核染色する。 14. 流水中で5分水洗する。 15. エタノールで脱水,キシロ一ルで透徹後,ビオライトまたはバルサムで封入する。 本染色法は従来の光顕免疫染色法と比較すると反応の増強性,特異性,感度ならびに反応産物の可視性が優れた染色方法であり,染色操作も簡便で,使用する一次抗体およびPAGを高希釈で用いるため経済性の高い有用な方法である。さらに大きな特徴として,プロティンAが数種の動物のIgG-Fcフラグメントに結合する能力を有するので染色に用いる一次抗体の作成動物を一種に限定しないという利点があげられ,また本染色法と酵素抗体法とを組み合わせて既存の方法より優れた二重染色法も可能である。以上,本研究で開発したPAGS染色法は多くの優れた特徴を有する染色法であり,光顕免疫染色法として普遍的に活用し得る技術であると考えられる。

Since the advent of colloidal gold as a marker particle for immunoelectron microscopy, a series of studies using the gold marker has successfully been made for solving various problems in electron microscopic histo- and cytochemistry. As a result of these studies, it has been revealed that colloidal gold particles can be conjugated to a variety of macromolecules such as immunoglobulins, enzymes, lectins, various proteins and polysaccharides to form stable complexes, and that those substances reacting with such complexes can be visualized by virtue of the presence of gold. The immunohistochemical methods employing gold-labeled immunogloblins are, however, subjected to the serious limitation that immunoglobulins of all animal species will not necessarily bind firmly to colloidal gold. To circumvent such a limitation, an indirect immunohistochemical method named the protein A gold technique has been developed, in which a protein A-colloidal gold complex was used as the second step reagent. Protein A, a major cell wall component of Staphylococcus aureus, shows a strong affinity for the Fc fragment of immunoglobulin G among different animal species. Most of the immunohistochemical techniques employing protein A gold have been developed for electron microscopy, and Roth was the first to use protein A gold for light microscopy and obtained pale reddish reaction products in pancreatic islets of the rat. The light microscopic protein A gold technique developed by Roth involved, however, at least a couple of difficulties ; in his technique, higher concentrations of primary antibodies and protein A gold were needed and the final reaction products obtained were not contrastive enough to be clearly distinguished from the background. To overcome these problems, an attempt has been made to photochemically intensify the reaction products obtained by the protein A gold technique using antibody against insulin in rat insular tissues and a protein A gold-silver method was successfully developed in which a refined procedure of physical development was performed. In this protein A gold-silver technique of Fujimori and Nakamura, however, high concentrations of primary antibody and protein A gold were employed. To establish a more sensitive and efficient protein A gold-silver staining method, in the present study, the availability of the staining technique was estimated in the course of the immunohistochemical demonstration of insular hormones in rat, dog, monkey and human pancreas. Pieces of tissues from the splenic portion of rat, dog, monkey and human pancreas were fixed in Bouin's fixative at 4°C for 18 hours, dehydrated in a graded ethanol series, cleared in benzene and embedded in paraffin wax. Sections were cut at a thickness of 4 μm and mounted on uncoated glass slides. Guinea pig antiserum against porcine insulin and rabbit antisera against porcine glucagon, somatostatin and against human pancreatic polypeptide were employed as the primary antibody. For the second step reagents of protein A gold-silver staining, protein A gold with particle sizes of both 5 nm and 10 nm were used. As reagents for the physical development combined with the immunohistochemical procedures, gum arabic, silver nitrate, citric acid and bromohydroquinone were employed. In the protein A gold-silver staining procedure previously developed by the author and co-worker, primary antibody against insulin and protein A gold employed were diluted 1:400 and 1:40 respectively and a gold particle size of 10 nm was specified. Based upon the results obtained by the previous procedure, attempts have been made to elevate sensitivity of the previous procedure by diluting primary antibody against insulin up to 1:10,000 and protein A gold up to 1:640. For further elevation of sensitivity of the staining procedure, protein A gold of a smaller particle size (5 nm) was used instead of 10 nm particles. To substantiate the wide applicability of the protein A gold-silver procedure to a variety of tissue antigens in light microscopy, the procedure was likewise employed for the immunohistochemical detection of three other endocrine pancreatic hormones, glucagon, somatostatin and pancreatic polypeptide (PP). In the course of this, the primary antibody against each hormone and protein A gold were investigated in terms of concentrations, and the gold particle sizes of both 5 and 10 nm were tested, as in the case with insulin. For substantiating the applicability of the staining procedure, in addition, double immunostaining has been examined to combine the protein A gold-silver staining method for glucagon with the immunoperoxidase technique for insulin. The formula of the developing solution employed is as follows: Solution A: 20% gum arabic aqueous solution, 45 ml; 10% silver nitrate aqueous solution, 1 ml. The gum arabic solution was prepared by centrifugation at 18,000 rpm for 30 min at 0℃ and resultant supernatant fluid was separated for use. Solution B: distilled water, 15 ml; bromohydroquinone, 200 mg; citric acid, 300 mg. The working developing solution was prepared by mixing solutions A and B immediately prior to use. The results obtained by the procedure of physical development may immediately be checked microscopically either in an open laboratory or in a dark room under a photographic safelight illumination. To compare the potency of this developer with that of different developers described previously, the former was replaced by the latter in the protein A gold-silver staining method. The developers compared were those of Danscher and of Moeremans et al. which contained silver lactate and hydroquinoe as developing reagents. To test the specificity of the staining reactions, four types of control procedures were performed : (1) incubation with the antigen-preabsorbed antibodies at concentration of 1 mg (insulin, glucagon and somatostatin) or 1μg (PP) antigen per 100μl (anti-insulin, -glucagon and -somatostatin) or 10μl (anti-PP) of undiluted antibody, followed by treatment with protein A gold solution and physical development; (2) incubation with the specific antibodies, followed by 1 hour incubation with unlabeled protein A (1 mg/1 ml), then with protein A gold solution and finally with physical developer; (3) omission of incubation with both antibodies and protein A gold solutions followed by physical development and (4) omission of physical development only. As compared with the previous technique, the protein A gold-silver staining procedure developed in the present study was found to show a significantly higher sensitivity. In view of the final staining results obtained, the dilutions of 1:2,000 for primary antibody against insulin and of 1:80 for protein A gold were found to be most appropriate in the staining with protein A gold of 10 nm particle size. In the protein A gold-silver procedure employing 5 nm particle size, the most optimal dilutions of primary antibody against insulin and of protein A gold were shown to be 1:10,000 and 1:320 respectively. Further, the most appropriate concentrations determined similarly for the primary antibodies against three other insular hormones and protein A gold in each case are shown as follows.(Chart) In the present study, the following staining protocols were established as a standard technique.(1) Deparaffinize sections and hydrate them in an ethanol series of descending concentrations.(2) Rinse for 15 min. in 3 changes of phosphate buffered saline (PBS : 0.01 M, pH 7.4).(3) Immerse for 30 min. in 5 % ovalbumin in PBS.(4) Incubate for 60-90 min. with a primary antibody diluted in PBS containing 1% BSA (BSA-PBA).(5) Rinse for 30 min. in 4 changes of PBS.(6) Immerse for 15 min. in 5 % ovalbumin in PBS.(7) Incubate for 60 min. with protein A gold diluted in BSA-PBS.(8) Rinse for 30 min. in 4 changes of phosphate buffer (0.01 M, pH 7.4).(9) Place in the physical developer (Fujimori and Nakamura 1985) for 60-80 min. at 20℃ either in a dark box or in a dark room.(10) Wash for 5 min. in running tap water and then immerse for 1 min. in a photographic fixer diluted 1:4.(11) Wash for 10 min. in running tap water.(12) Counterstain with Kernechtrot.(13) Rinse in water.(14) Dehydrate in a graded ethanol series, clear in xylene and mount in permanent media such as Canada balsam. In the endocrine pancreatic tissues from the four mammalian species immumostained for insulin, glucagon, somatostatin and pancreatic polypeptide by means of the present procedure, reaction products were visualized in black shades. Higher magnifications of the cytoplasm of each endocrine cell type revealed that these products consisted of distinct fine particles of different sizes. In the present procedure, the staining images at both low and high magnifications obtained with gold particle size of 5 nm were comparable in quality to those obtained by with gold particle size of 10 nm. In the present double staining procedure, two types of endocrine cells secreting glucagon and insulin respectively have simultaneously been visualized by reaction products of different shades in an identical pancreatic islet, i.e. reaction products obtained by the protein A gold-silver technique were revealed in black shades, whereas those obtained by the immunoperoxidase method were visualized in brown colors. In the protein A gold-silver staning procedure employing Danscher developer, weak reaction products were obtained as pale black shades. Contrary to this, the use of Moeremans et al. developer resulted in abolishment of all immunostaining. Negative results were constantly obtained, whenever sections were subjected to the control staining procedures (1),(2),(3) and (4). In the present protein A gold-silver staining procedure employing gold particle size of 10 nm, the higher dilutions of primary antibodies were found to result in a higher efficiency of the procedure. Moreover, the present staining procedure with smaller gold particle size of 5 nm revealed a higher sensitivity, as compared with that using 10 nm gold particle size, and this has made possible further dilution of primary antibodies and protein A gold. In the present staining procedure employing 5 nm gold particles, a longer duration of development was needed so as to obtain satisfactory results, as compared with that using 10 nm gold particles. This appears to reflect that a longer time lapse is necessary for the visualization of silver precipitates in light microscopy. A higher dilution of primary antibodies and protein A gold and smaller particle size of protein A gold could induce time-consuming effects on the visualization of precipitates. In double stainings of immunohistochemistry, it is generally recognized that two antigenic sites are to be clearly distinguished without difficulty and unreliability. In the present double staining procedure, the black reaction products obtained by the protein A gold-silver staining method were highly contrastive to the brown ones obtained by the immunoperoxidase technique. In view of this, the present double staining procedure is believed to be superior to those previously developed such as immunoperoxidase and immunocolloid techniques, in terms of the sharp contrast between the colors exhibited by the two reaction products obtained, In light microscopy, attempts have hitherto been made to photochemically intensify the reaction products obtained by techniques employing gold-labeled RN-ase for RNA, immunogold for antigens, protein A gold for antigens, lectin-gold for glycoproteins and biotinylated lectin-avidin gold for glycoproteins. In all these techniques, except for the protein A gold-silver staining procedure, Danscher's developer or its modification such as that of Moeremans et al. were employed and these developers involved silver lactate and hydroquinone as developing reagents. On the contrary, an improved physical developer was employed in the protein A gold-silver staining procedure, which contained silver nitrate and bromohydroquinone as developing substances and ultracentrifugated gum arabic solution as protective colloid. Silver nitrate is known to be more potent in terms of silver ion supply, as compared with silver lactate. Bromohydroquinone exhibits a significantly higher developing capacity than hydroquinone. The purified gum arabic solution is effective for suppression of autocatlytic activity of the developer. These ingredients are thought to result in a potent developer, which can lead to the visualization of even a small amount of gold particles on antigenic sites in light microscopy. In view of all the results obtained by the present experimental and control stainings, the protein A gold-silver staining procedure established here is believed to be not only high in specificity and efficiency but marked in intensity. In addition, the present double staining method in combination with the immunoperoxidase method was conceived to be a reliable and useful technique for the simultaneous detection of two tissue antigens in light microscopic immunohistochemistry. As the results obtained indicate, the present staining procedure was found to be useful consistently for the detection of four pancreatic hormones from different mammalian species. Since protein A shows a strong affinity towards the Fc fragment of IgG from numerous animal species, furthermore, the present protein A gold-silver staining procedure can be widely applied to demonstrate a variety of tissue antigens. Protein A gold-silver techniuque is, therefore, a reliable method of choice in light microscopic immunohistochemistry.

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