JP5199220B2 - Electrostaining agent, staining solution, and electrostaining method - Google Patents
Electrostaining agent, staining solution, and electrostaining method Download PDFInfo
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- 239000012192 staining solution Substances 0.000 title claims description 21
- 238000000034 method Methods 0.000 title description 8
- 238000010186 staining Methods 0.000 claims description 50
- 230000005540 biological transmission Effects 0.000 claims description 27
- 239000003795 chemical substances by application Substances 0.000 claims description 26
- 239000012472 biological sample Substances 0.000 claims description 22
- JPDBEEUPLFWHAJ-UHFFFAOYSA-K samarium(3+);triacetate Chemical group [Sm+3].CC([O-])=O.CC([O-])=O.CC([O-])=O JPDBEEUPLFWHAJ-UHFFFAOYSA-K 0.000 claims description 20
- LYQGMALGKYWNIU-UHFFFAOYSA-K gadolinium(3+);triacetate Chemical compound [Gd+3].CC([O-])=O.CC([O-])=O.CC([O-])=O LYQGMALGKYWNIU-UHFFFAOYSA-K 0.000 claims description 14
- 239000007864 aqueous solution Substances 0.000 claims description 12
- 238000007447 staining method Methods 0.000 claims description 10
- 150000003839 salts Chemical class 0.000 claims description 9
- 229910052688 Gadolinium Inorganic materials 0.000 claims description 6
- 229910052772 Samarium Inorganic materials 0.000 claims description 6
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 claims description 6
- KZUNJOHGWZRPMI-UHFFFAOYSA-N samarium atom Chemical compound [Sm] KZUNJOHGWZRPMI-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical group CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical class Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 2
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 24
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 21
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- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 229910052802 copper Inorganic materials 0.000 description 6
- 239000010949 copper Substances 0.000 description 6
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- 239000012528 membrane Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
- 108010064539 amyloid beta-protein (1-42) Proteins 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
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- 238000010894 electron beam technology Methods 0.000 description 3
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- 229910052762 osmium Inorganic materials 0.000 description 3
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 3
- 229920000647 polyepoxide Polymers 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- MEANOSLIBWSCIT-UHFFFAOYSA-K gadolinium trichloride Chemical compound Cl[Gd](Cl)Cl MEANOSLIBWSCIT-UHFFFAOYSA-K 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- XZVBUSAPGMMCSW-UHFFFAOYSA-L 2-amino-3-methyl-4h-imidazol-5-one;dichloroplatinum Chemical compound Cl[Pt]Cl.CN1CC(=O)N=C1N.CN1CC(=O)N=C1N XZVBUSAPGMMCSW-UHFFFAOYSA-L 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
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- RGCKGOZRHPZPFP-UHFFFAOYSA-N Alizarin Natural products C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- HFVAFDPGUJEFBQ-UHFFFAOYSA-M alizarin red S Chemical compound [Na+].O=C1C2=CC=CC=C2C(=O)C2=C1C=C(S([O-])(=O)=O)C(O)=C2O HFVAFDPGUJEFBQ-UHFFFAOYSA-M 0.000 description 1
- 108010064397 amyloid beta-protein (1-40) Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000003758 nuclear fuel Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- KKCBUQHMOMHUOY-UHFFFAOYSA-N sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 description 1
- 229910001948 sodium oxide Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
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- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
本発明は、電子染色剤、染色液および電子染色方法に関し、特に透過型電子顕微鏡を用いて観察される生体試料に好適なものに関する。 The present invention relates to an electron staining agent, a staining solution, and an electron staining method, and particularly relates to a material suitable for a biological sample observed using a transmission electron microscope.
生体試料は炭素、酸素、窒素、および水素などの軽元素で構成される。このため、透過型電子顕微鏡を用いて生体試料の構造を観察するには、通常、電子線を散乱する物質を結合させる電子染色が必要である。このような電子染色に用いられる酢酸ウラニルは性能、使用頻度ともに最も高い。 The biological sample is composed of light elements such as carbon, oxygen, nitrogen, and hydrogen. For this reason, in order to observe the structure of a biological sample using a transmission electron microscope, electron staining that binds a substance that scatters an electron beam is usually required. Uranyl acetate used for such electron staining has the highest performance and frequency of use.
ところが、酢酸ウラニルは「核燃料」であるため、とりわけ我が国においては、その入手や搬送法、使用法、廃棄法に至るまでさまざまな形で規制が成されてきた。近年の世界情勢に鑑み規制はさらに厳しく強化される方向にあるので、一般の研究者が必要な時に必要な量の酢酸ウラニルを自由に用いることは、よりいっそう困難となる。 However, since uranyl acetate is a “nuclear fuel,” especially in Japan, regulations have been established in various forms ranging from acquisition, transportation, usage, and disposal. In view of the recent global situation, regulations are becoming stricter, and it is even more difficult for general researchers to freely use the necessary amount of uranyl acetate when needed.
この問題を解決するため、酢酸ウラニルの代替品としてアリザリンや、シスプラチンを基本とする白金ブルーなどが提案され、一部で市販もされている（例えば、特許文献１）。 In order to solve this problem, alizarin or platinum blue based on cisplatin has been proposed as an alternative to uranyl acetate, and some of them are commercially available (for example, Patent Document 1).
上記酢酸ウラニルの代替品は主に樹脂包埋切片の電子染色剤として用いられるが、安定性に乏しいため使用前の調製が必要である上、その操作も面倒で使い勝手が好いとは言い難い。また、ネガティブ染色用の電子染色剤としては燐タングステン酸ナトリウムなども使用されるが固定作用は弱く、染色や固定など酢酸ウラニルの全ての使途を代替できる電子染色剤は、未だ見出されていない。 The above-mentioned uranyl acetate substitute is mainly used as an electronic stain for resin-embedded sections. However, since it is poor in stability, preparation before use is necessary, and the operation is cumbersome and difficult to use. In addition, sodium phosphotungstate is used as an electron stain for negative staining, but its fixing action is weak, and no electronic stain that can replace all uses of uranyl acetate such as staining and fixation has yet been found. .
そこで本発明は、透過型電子顕微鏡で生体試料を観察するための高性能かつ、安全で、容易に取り扱うことができる電子染色剤、染色液および電子染色方法を提供することを目的とする。 Accordingly, an object of the present invention is to provide a high-performance, safe, and easy-to-handle electronic stain, staining solution, and electronic staining method for observing a biological sample with a transmission electron microscope.
本発明の請求項１に係る発明は、透過型電子顕微鏡で観察される生体試料の画像に濃淡をつけるための電子染色剤において、サマリウムまたはガドリニウムを含む塩であることを特徴とする。 The invention according to claim 1 of the present invention is an electron staining agent for imparting light and shade to an image of a biological sample observed with a transmission electron microscope, and is a salt containing samarium or gadolinium.
本発明の請求項２に係る発明は、前記塩が酢酸サマリウムまたは酢酸ガドリニウムであることを特徴とする。 The invention according to claim 2 of the present invention is characterized in that the salt is samarium acetate or gadolinium acetate.
本発明の請求項３に係る発明は、前記塩が酢酸塩または塩酸塩であることを特徴とする。
The invention according to claim 3 of the present invention, the salt is characterized in that is acetate or a hydrochloric acid salt.
本発明の請求項４に係る発明は、透過型電子顕微鏡で観察される生体試料の画像に濃淡をつけるための電子染色剤を含有する染色液において、前記電子染色剤がサマリウムまたはガドリニウムを含む塩であることを特徴とする。 The invention according to claim 4 of the present invention is a staining solution containing an electron staining agent for adding light and shade to an image of a biological sample observed with a transmission electron microscope, wherein the electron staining agent contains samarium or gadolinium. It is characterized by being.
本発明の請求項５に係る発明は、前記電子染色剤が酢酸サマリウムまたは酢酸ガドリニウムであって、前記電子染色剤を１質量％以上１０質量％以下含有する水溶液であることを特徴とする。 The invention according to claim 5 of the present invention is characterized in that the electron staining agent is samarium acetate or gadolinium acetate and is an aqueous solution containing 1% by mass to 10% by mass of the electron staining agent.
本発明の請求項６に係る発明は、電子染色剤を含有する染色液に生体試料を接触させ、透過型電子顕微鏡で観察される前記生体試料の画像に濃淡をつける電子染色方法において、前記電子染色剤がサマリウムまたはガドリニウムを含む塩であることを特徴とする。 According to a sixth aspect of the present invention, there is provided an electronic staining method in which a biological sample is brought into contact with a staining solution containing an electron stain, and the image of the biological sample observed with a transmission electron microscope is shaded. The staining agent is a salt containing samarium or gadolinium.
本発明の請求項７に係る発明は、前記電子染色剤が酢酸サマリウムまたは酢酸ガドリニウムであって、前記染色液が前記電子染色剤を１質量％以上１０質量％以下含有する水溶液であることを特徴とする。 The invention according to claim 7 of the present invention is characterized in that the electron staining agent is samarium acetate or gadolinium acetate, and the staining liquid is an aqueous solution containing 1% by mass to 10% by mass of the electron staining agent. And
本発明によれば、酢酸ウラニルとほぼ同等の染色および固定効果を得ることができ、酢酸ウラニルと比較して極めて安全で、容易に取り扱うことができる。 According to the present invention, it is possible to obtain the same staining and fixing effect as uranyl acetate, and it is extremely safe and easy to handle as compared to uranyl acetate.
本発明に係る染色液は、電子染色剤を含有する水溶液からなる。電子染色剤は、ランタノイド元素を含む化合物で構成される。前記化合物は、酢酸サマリウム、または酢酸ガドリニウムを選択することができる。本実施形態の場合、染色液は、電子染色剤を１質量％以上１０質量％以下含有することが好ましい。 The staining liquid according to the present invention comprises an aqueous solution containing an electronic staining agent. The electronic stain is composed of a compound containing a lanthanoid element. As the compound, samarium acetate or gadolinium acetate can be selected. In the case of the present embodiment, the staining solution preferably contains 1% by mass or more and 10% by mass or less of an electronic staining agent.
この電子染色剤は、生体試料を透過電子顕微鏡で観察するための前処理として、生体試料の固定および染色に用いられ、当該生体試料のネガティブ染色用の電子染色剤として、従来の酢酸ウラニルとほぼ同等の染色および固定効果を得ることができる。 This electron stain is used as a pretreatment for observing a biological sample with a transmission electron microscope, and is used for fixing and staining the biological sample. As an electron stain for negative staining of the biological sample, it is almost the same as conventional uranyl acetate. Equivalent staining and fixing effects can be obtained.
また、本発明に係る染色液は、酢酸サマリウム、または酢酸ガドリニウムを含有する水溶液で構成されているので、酢酸ウラニルと比較して極めて安全で、容易に取り扱うことができる。 Further, since the staining liquid according to the present invention is composed of an aqueous solution containing samarium acetate or gadolinium acetate, it is extremely safe and can be handled easily compared to uranyl acetate.
因みに、従来、樹脂包埋した組織・細胞試料の切片に対して行う電子染色は、酢酸ウラニルとクエン酸鉛（佐藤氏鉛液など）による２重染色が行われる。 Incidentally, conventionally, the electronic staining performed on a section of a tissue / cell sample embedded in a resin is performed by double staining with uranyl acetate and lead citrate (such as Mr. Sato's lead solution).
これに対し、本発明に係る電子染色剤は、単独で十分な染色効果を示し、クエン酸鉛による処理を省略することができる。これにより極めて簡易に電子染色を行うことができる。 On the other hand, the electronic stain according to the present invention exhibits a sufficient staining effect by itself, and the treatment with lead citrate can be omitted. As a result, electronic staining can be performed very easily.
また、本発明に係る電子染色剤は、蛋白質複合体の固定効果を利用して回転シャドウィング・レプリカ法の前処理剤としても用いることができる。回転シャドウィング・レプリカ法とは、対象分子を回転しながらその表面に重金属微粒子を蒸着して、そのカーボン・レプリカを電子顕微鏡観察する方法である。 Further, the electron staining agent according to the present invention can be used as a pretreatment agent for the rotational shadowing replica method by utilizing the fixing effect of the protein complex. The rotational shadowing replica method is a method in which heavy metal fine particles are deposited on the surface of a target molecule while rotating the target molecule, and the carbon replica is observed with an electron microscope.
次に、本発明に係る電子染色剤を用いたネガティブ染色法の概要を説明する。なお、染色方法は対象とする生体試料の種類などにより多様であり、以下の記載に限定されるものではない。 Next, an outline of the negative staining method using the electronic staining agent according to the present invention will be described. The staining method varies depending on the type of the biological sample to be processed, and is not limited to the following description.
まず、生体試料を含有する試料溶液をグリッド上の支持膜に載せ、所定時間静置して吸着させた後、試料溶液とほぼ同量の本発明に係る染色液に接触させるか、あるいは数滴の染色液を直接滴下する。接触時間は、１〜４０秒が好ましい。次いで、余剰の染色液を濾紙で吸収してから自然乾燥させると、電子染色剤が蛋白質複合体などの試料の間隙や周囲に残留する。そのように処理された生体試料（以下、「観察試料」という）を透過型電子顕微鏡で観察すると、電子染色剤が残留した部分では電子線が強く散乱され、観察試料の凸部は電子線が透過するため、対象は縁取りされた輪郭中に明るく浮き上がって見える（ネガティブ染色）。電子染色剤はこのように、生体試料の凹凸に応じた濃淡画像を呈することができる。 First, a sample solution containing a biological sample is placed on a support membrane on a grid and allowed to stand for a predetermined time to be adsorbed, and then contacted with the same amount of the staining solution according to the present invention as the sample solution, or a few drops Directly drop the staining solution. The contact time is preferably 1 to 40 seconds. Next, when the excess staining solution is absorbed with a filter paper and then naturally dried, the electron staining agent remains in the gap or the periphery of the sample such as the protein complex. When the biological sample thus treated (hereinafter referred to as “observation sample”) is observed with a transmission electron microscope, the electron beam is strongly scattered in the portion where the electron stain remains, and the electron beam is observed on the convex portion of the observation sample. Because of transmission, the object appears brightly floating in the outlined outline (negative staining). Thus, the electronic stain can present a grayscale image corresponding to the unevenness of the biological sample.
また、本発明に係る電子染色剤は、上記の染色方法の他、樹脂包埋の後に薄切した組織の染色、並びに蛋白質試料の固定などにも用いることができる。
（実施例）
（実施例１）
支持膜としてフォルムバールを張った銅グリッド上に、試料溶液としてアミロイドβ４２線維溶液を３０秒間静置後、過剰溶液を濾紙で除去した。次いで、染色液として５μｌの２．５％酢酸サマリウム水溶液を加えて１０秒間静置後、水分を濾紙にて除去し、室温で乾燥してから透過電子顕微鏡を用いて像を観察した。その結果を図１に示す。後述する比較例１と比較して、酢酸サマリウムは、酢酸ウラニルとほぼ同等の染色および固定効果が得られることを確認した。
（実施例２）
支持膜としてフォルムバールを張った銅グリッド上に、試料溶液としてアミロイドβ４２線維溶液を３０秒間静置後、過剰溶液を濾紙で除去した。次いで、染色液として５μｌの２．５％酢酸ガドリニウム水溶液を加えて１０秒間静置後、水分を濾紙にて除去し、室温で乾燥してから透過電子顕微鏡を用いて像を観察した。その結果を図２に示す。後述する比較例１と比較して、酢酸ガドリニウムは、酢酸ウラニルとほぼ同等の染色および固定効果が得られることを確認した。
（実施例３）
支持膜としてフォルムバールを張った銅グリッド上に、試料溶液としてアミロイドβ４２線維溶液を３０秒間静置後、過剰溶液を濾紙で除去した。次いで、染色液として５μｌの２．５％塩化ガドリニウム水溶液を加えて１０秒間静置後、水分を濾紙にて除去し、室温で乾燥してから透過電子顕微鏡を用いて像を観察した。その結果を図３に示す。後述する比較例１と比較して、塩化ガドリニウムは、酢酸ウラニルとほぼ同等の染色および固定効果が得られることを確認した。
（参考例１）
生体試料として２％グルタルアルデヒドと２％オスミウムで化学固定したマウス肝臓をエポキシ樹脂に包埋後、ウルトラミクロトームで８０ｎｍに薄切してグリッドに載せた。パラフィルム上に染色液として１０％ 酢酸サマリウム １０μｌの水滴を置き、グリッドの切片側を接触させて２０分間染色した。次いで、同様にパラフィルムに載せた蒸留水５０μｌの水滴で１０秒間洗浄した。同一操作を３回繰り返した後、乾燥して電子顕微鏡観察を行った。その結果を図４に示す。後述する比較例２と比較して、酢酸サマリウムは、酢酸ウラニルと同様に、樹脂包埋の後に薄切した組織の染色に使用できることを確認した。
（参考例２）
生体試料として２％グルタルアルデヒドと２％オスミウムで化学固定したマウス肝臓をエポキシ樹脂に包埋後、ウルトラミクロトームで８０ｎｍに薄切してグリッドに載せた。パラフィルム上に染色液として１０％ 酢酸ガドリニウム １０μｌの水滴を置き、グリッドの切片側を接触させて２０分間染色した。次いで、同様にパラフィルムに載せた蒸留水５０μｌの水滴で１０秒間洗浄した。同一操作を３回繰り返した後、乾燥して電子顕微鏡観察を行った。その結果を図５に示す。後述する比較例２と比較して、酢酸ガドリニウムは、酢酸ウラニルと同様に、樹脂包埋の後に薄切した組織の染色に使用できることを確認した。
（参考例３ 回転シャドイウィング・レプリカ法）
試料溶液としてアミロイドβ４０線維水溶液２００μｌを雲母板に載せ、４０秒間静置後、蒸留水５００μｌで洗浄した。雲母板を ０．１Ｍ 酢酸アンモニウム・３０％グリセロール ５００μｌで２回、さらに酢酸サマリウム２００ｍｇ、蒸留水 ３．５ｍｌ、グリセロール １．５ｍｌの混液に接触させ、染色液として酢酸サマリウム２００ｍｇ、蒸留水 ３．５ｍｌ、グリセロール １．５ｍｌの混液で ４０秒間固定してから、０．１Ｍ 酢酸アンモニウム、３０％グリセロール混液 ５００μｌにより３回洗浄した。次いで、同一形状の雲母板を試料吸着面に強く圧着して残留溶液を除去した。その雲母板を真空中に設置して白金・カーボンを回転蒸着後、カーボン膜で補強した。蒸留水の表面張力で雲母板から外したレプリカ膜を銅グリッドに載せ、透過電子顕微鏡により観察した。その結果を図６に示す。酢酸サマリウムは、蛋白質複合体の固定効果を利用して回転シャドウィング・レプリカ法の前処理剤としても使用できることを確認した。
（参考例４ 回転シャドイウィング・レプリカ法）
５ｍＭ 塩化マグネシウム、１０ｍＭ 塩化カリウム、１０ ｍＭ ［２−［４−（２−ヒドロキシエチル）−１−ピペラジニル］エタンスルホン酸・水酸化ナトリウム緩衝液（ｐＨ８）中で重合させた試料溶液としてのアクチン繊維溶液 ５０μｌを雲母板に載せ、４０秒間静置後、０．１％グルタールアルデヒドを添加した １０ ｍＭ Ｍ−［２−［４−（２−ヒドロキシエチル）−１−ピペラジニル］エタンスルホン酸・水酸化ナトリウム緩衝液（ｐＨ８）５００μｌに接触させ、０．１Ｍ 酢酸アンモニム、３０％グリセロール溶液 ５００μｌで２回、染色液として酢酸サマリウム２５０ｍｇ蒸留水３．５ｍｌ、グリセロール１．５ｍｌを含有する ３０％グリセロール混液 ５００μｌで３回洗浄した。次いで、同一形状の雲母板を強く圧着して残留溶液を除去した。雲母板を真空中に設置して白金・カーボンを回転蒸着後、カーボン膜で補強した。蒸留水の表面張力で雲母板から外したレプリカ膜を銅グリッドに載せ、透過電子顕微鏡により観察した。その結果を図７に示す。酢酸サマリウムは、蛋白質複合体の固定効果を利用して回転シャドウィング・レプリカ法の前処理剤としても使用できることを確認した。
（比較例１）
支持膜としてフォルムバールを張った銅グリッド上にアミロイドβ４２線維溶液を３０秒間静置後、過剰溶液を濾紙で除去した。次いで、５μｌの ４％酢酸ウラニル水溶液を加えて１０秒間静放置後、水分を濾紙にて除去し、室温で乾燥してから透過電子顕微鏡を用いて像を観察した（図８）。
（比較例２）
２％グルタルアルデヒドと２％オスミウムで化学固定したマウス肝臓をエポキシ樹脂に包埋後、ウルトラミクロトームで８０ｎｍに薄切してグリッドに載せた。パラフィルム上に ４％酢酸ウラニル １０μｌの水滴を置き、グリッドの切片側を接触させて２０分間、さらに佐藤氏鉛溶液で１０分間の後染色を施した。次いで、同様にパラフィルムに載せた蒸留水５０μｌの水滴で１０秒間洗浄した。同一操作を３回繰り返した後、乾燥して電子顕微鏡観察を行った（図９）。
（変形例）
本発明は上記実施形態に限定されるものではなく、本発明の趣旨の範囲内で適宜変更することが可能である。例えば、電子染色は、生体試料を電子染色剤に所定時間、浸漬することによるブロック染色にて行ってもよい。
In addition to the staining method described above, the electronic stain according to the present invention can also be used for staining a tissue sliced after embedding a resin and fixing a protein sample.
(Example)
Example 1
An amyloid β42 fiber solution was allowed to stand as a sample solution for 30 seconds on a copper grid coated with form bar as a support membrane, and then the excess solution was removed with filter paper. Next, 5 μl of a 2.5% samarium acetate aqueous solution was added as a staining solution and allowed to stand for 10 seconds, after which water was removed with a filter paper and dried at room temperature, and then the image was observed using a transmission electron microscope. The result is shown in FIG. As compared with Comparative Example 1 described later, it was confirmed that samarium acetate can obtain substantially the same staining and fixing effect as uranyl acetate.
(Example 2)
An amyloid β42 fiber solution was allowed to stand as a sample solution for 30 seconds on a copper grid coated with form bar as a support membrane, and then the excess solution was removed with filter paper. Next, 5 μl of a 2.5% gadolinium acetate aqueous solution was added as a staining solution and allowed to stand for 10 seconds. Then, moisture was removed with a filter paper, dried at room temperature, and an image was observed using a transmission electron microscope. The result is shown in FIG. Compared to Comparative Example 1 described later, it was confirmed that gadolinium acetate can obtain substantially the same staining and fixing effect as uranyl acetate.
(Example 3)
An amyloid β42 fiber solution was allowed to stand as a sample solution for 30 seconds on a copper grid coated with form bar as a support membrane, and then the excess solution was removed with filter paper. Next, 5 μl of a 2.5% gadolinium chloride aqueous solution was added as a staining solution and allowed to stand for 10 seconds. Then, moisture was removed with a filter paper, dried at room temperature, and an image was observed using a transmission electron microscope. The result is shown in FIG. Compared to Comparative Example 1 described later, it was confirmed that gadolinium chloride can obtain substantially the same staining and fixing effect as uranyl acetate.
( Reference Example 1 )
A mouse liver chemically fixed with 2% glutaraldehyde and 2% osmium as a biological sample was embedded in an epoxy resin, sliced to 80 nm with an ultramicrotome, and placed on a grid. A 10 μl drop of 10% samarium acetate as a staining solution was placed on the parafilm, and the section side of the grid was contacted to stain for 20 minutes. Subsequently, it was washed for 10 seconds with a water droplet of 50 μl of distilled water similarly placed on parafilm. The same operation was repeated three times, then dried and observed with an electron microscope. The result is shown in FIG. As compared with Comparative Example 2 described later, it was confirmed that samarium acetate can be used for staining of sliced tissue after resin embedding, like uranyl acetate.
( Reference Example 2 )
A mouse liver chemically fixed with 2% glutaraldehyde and 2% osmium as a biological sample was embedded in an epoxy resin, sliced to 80 nm with an ultramicrotome, and placed on a grid. 10 μl of 10% gadolinium acetate as a staining solution was placed on the parafilm, and the section side of the grid was contacted to stain for 20 minutes. Subsequently, it was washed for 10 seconds with a water droplet of 50 μl of distilled water similarly placed on parafilm. The same operation was repeated three times, then dried and observed with an electron microscope. The result is shown in FIG. Compared to Comparative Example 2 described below, it was confirmed that gadolinium acetate can be used for staining of sliced tissue after resin embedding, like uranyl acetate.
( Reference Example 3 Rotating Shadow Ewing Replica Method)
As a sample solution, 200 μl of an amyloid β40 fiber aqueous solution was placed on a mica plate, allowed to stand for 40 seconds, and then washed with 500 μl of distilled water. The mica plate was contacted twice with 500 μl of 0.1 M ammonium acetate / 30% glycerol, and further contacted with a mixed solution of 200 mg of samarium acetate, 3.5 ml of distilled water and 1.5 ml of glycerol, and 200 mg of samarium acetate as a staining solution and 3.5 ml of distilled water. Then, the mixture was fixed with 1.5 ml of glycerol for 40 seconds, and then washed three times with 500 μl of 0.1 M ammonium acetate / 30% glycerol. Next, the mica plate having the same shape was strongly pressed against the sample adsorption surface to remove the residual solution. The mica plate was placed in a vacuum, and platinum and carbon were rotoevaporated and then reinforced with a carbon film. The replica film removed from the mica plate by the surface tension of distilled water was placed on a copper grid and observed with a transmission electron microscope. The result is shown in FIG. It was confirmed that samarium acetate can also be used as a pretreatment agent for the rotational shadowing replica method by utilizing the fixing effect of the protein complex.
( Reference Example 4 Rotating Shadow Ewing Replica Method)
Actin fiber as a sample solution polymerized in 5 mM magnesium chloride, 10 mM potassium chloride, 10 mM [2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid / sodium hydroxide buffer (pH 8) 50 μl of the solution was placed on a mica plate, allowed to stand for 40 seconds, and then added with 0.1% glutaraldehyde. 10 mM M- [2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid / water Contact with 500 μl of sodium oxide buffer (pH 8), twice with 500 μl of 0.1 M ammonium acetate, 30% glycerol solution, 30% glycerol mixture containing 3.5 ml of samarium acetate 250 mg distilled water and 1.5 ml of glycerol as staining solution Washed 3 times with 500 μl. Subsequently, the mica plate having the same shape was strongly pressed to remove the residual solution. A mica plate was placed in a vacuum, and platinum and carbon were rotoevaporated and then reinforced with a carbon film. The replica film removed from the mica plate by the surface tension of distilled water was placed on a copper grid and observed with a transmission electron microscope. The result is shown in FIG. It was confirmed that samarium acetate can also be used as a pretreatment agent for the rotational shadowing replica method by utilizing the fixing effect of the protein complex.
(Comparative Example 1)
The amyloid β42 fiber solution was allowed to stand for 30 seconds on a copper grid with a form bar as a supporting membrane, and then the excess solution was removed with filter paper. Next, 5 μl of a 4% uranyl acetate aqueous solution was added and allowed to stand for 10 seconds, after which moisture was removed with a filter paper, dried at room temperature, and an image was observed using a transmission electron microscope (FIG. 8).
(Comparative Example 2)
Mouse liver chemically fixed with 2% glutaraldehyde and 2% osmium was embedded in an epoxy resin, sliced to 80 nm with an ultramicrotome, and placed on a grid. A water droplet of 10 μl of 4% uranyl acetate was placed on the parafilm, and the section side of the grid was contacted for 20 minutes, followed by post-staining with Mr. Sato's lead solution for 10 minutes. Subsequently, it was washed for 10 seconds with a water droplet of 50 μl of distilled water similarly placed on parafilm. The same operation was repeated three times, and then dried and observed with an electron microscope (FIG. 9).
(Modification)
The present invention is not limited to the above-described embodiment, and can be appropriately changed within the scope of the gist of the present invention. For example, the electronic staining may be performed by block staining by immersing a biological sample in an electronic staining agent for a predetermined time.
酢酸サマリウムおよび酢酸ガドリニウムの強い毒性は報告されていない。酢酸サマリウムの濃厚溶液に皮膚が触れて軽度な炎症を起こした例はあるが、酢酸ガドリニウムでは、濃厚溶液でもそれさえ起こさず、強い毒性を示す酢酸ウラニルと比較して極めて安全性の高い物質である。そのため入手にも取り扱いにも制限がなく、溶液状態で安定であり保存も容易である。酢酸サマリウム水溶液は、その優れた特性により酢酸ウラニルの問題点を解消するとともに、それに代わる電子染色剤として、広範な使途が期待できる。 The strong toxicity of samarium acetate and gadolinium acetate has not been reported. There is an example of mild irritation caused by skin contact with a concentrated solution of samarium acetate, but gadolinium acetate does not even cause it to occur, and it is a highly safe substance compared to the highly toxic uranyl acetate. is there. For this reason, there is no restriction on the availability and handling, and it is stable in a solution state and easy to store. The aqueous solution of samarium acetate eliminates the problems of uranyl acetate due to its excellent properties and can be expected to be used widely as an electronic staining agent instead.
Claims (7)
前記電子染色剤がサマリウムまたはガドリニウムを含む塩であることを特徴とするネガティブ染色法のための染色液。 In a staining solution containing an electron staining agent for adding light and shade to an image of a biological sample observed with a transmission electron microscope,
A staining solution for negative staining, wherein the electron staining agent is a salt containing samarium or gadolinium.
前記電子染色剤を１質量％以上１０質量％以下含有する水溶液であることを特徴とする請求項４記載の染色液。 The electron stain is samarium acetate or gadolinium acetate,
The staining solution according to claim 4, wherein the staining solution is an aqueous solution containing 1% by mass or more and 10% by mass or less of the electron staining agent.
前記電子染色剤がサマリウムまたはガドリニウムを含む塩であることを特徴とするネガティブ染色法による電子染色方法。 In an electronic staining method in which a biological sample is brought into contact with a staining solution containing an electronic staining agent, and the image of the biological sample observed with a transmission electron microscope is shaded,
The electron staining method according to the negative staining method, wherein the electron staining agent is a salt containing samarium or gadolinium.
前記染色液が前記電子染色剤を１質量％以上１０質量％以下含有する水溶液であることを特徴とする請求項６記載の電子染色方法。
The electron stain is samarium acetate or gadolinium acetate,
The electronic staining method according to claim 6, wherein the staining liquid is an aqueous solution containing 1 to 10% by mass of the electronic staining agent.
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