JP2006266755A - Pretreatment liquid of slice of biotissue, and observation method of protein and nucleic acid in biotissue using it - Google Patents
Pretreatment liquid of slice of biotissue, and observation method of protein and nucleic acid in biotissue using it Download PDFInfo
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Abstract
Description
本発明は、生体組織切片の前処理液及びこれを用いた生体組織中の核酸及び蛋白質の観測方法に関する。 The present invention relates to a pretreatment liquid for a biological tissue section and a method for observing nucleic acids and proteins in the biological tissue using the same.
生体組織中の種々の物質、例えば、核酸や蛋白質の動態を観測するためには、生体組織をホルマリン等の固定液で固定し、エタノール等で脱水後、パラフィン包埋し、ミクロトームで組織を５μｍ程度の厚さに薄切りし、スライドグラスに載せ、脱パラフィン後、水洗し、目的に合わせて前処理し、変性処理を行い、標識プローブを用いたハイブリダイゼーションや標識抗体を用いた抗原抗体反応等を行い蛍光顕微鏡や共焦点レーザー顕微鏡を用いて観察し、目的の核酸や蛋白質の細胞内における位置情報を観測する。
前処理は細胞内の標的物質を露出させ、外部から供給する反応物質との反応を促進するための処理である。従来このような前処理液としては、緩衝液、例えば、リン酸緩衝液、クエン酸緩衝液等が用いられ、あるいは水を用いてオートクレーブ中で加熱加圧処理する方法等が用いられている。このような前処理液として、例えば、特許文献１には、２−メチルマレイン酸無水物（Citraconic anhydride)(CCA)を含有する抗原賦活剤が開示されている。
In order to observe the dynamics of various substances such as nucleic acids and proteins in living tissue, the living tissue is fixed with a fixative such as formalin, dehydrated with ethanol, embedded in paraffin, and the tissue is 5 μm with a microtome. Cut into thin sections, put on a slide glass, deparaffinized, washed with water, pretreated according to the purpose, denatured, hybridization using labeled probe, antigen-antibody reaction using labeled antibody, etc. And observe using a fluorescence microscope or a confocal laser microscope to observe the positional information of the target nucleic acid or protein in the cell.
The pretreatment is a treatment for exposing the target substance in the cell and promoting the reaction with the reactant supplied from the outside. Conventionally, as such a pretreatment solution, a buffer solution, for example, a phosphate buffer solution, a citrate buffer solution, or the like is used, or a method of heating and pressurizing in an autoclave using water is used. As such a pretreatment liquid, for example, Patent Document 1 discloses an antigen activator containing 2-methylmaleic anhydride (CCA).
蛋白質を観測する際の前処理液としては、クエン酸緩衝液やこれに界面活性剤を添加したものが使用されている。クエン酸緩衝液は熱処理の際に蛋白質が損傷を受けるのを防止する機能がある。また界面活性剤は細胞内の標的蛋白質を露出させ、外部から供給する反応物質との接触の機会を向上させる機能がある。しかし、オートクレーブの使用は危険を伴い、スライドグラスが割れたり、時間がかかるという問題の他、熱をかけすぎると細胞内の蛋白質の位置が乱れ、画像情報が壊れてしまうという問題がある。
また、核酸を観測する際の前処理液としては、蛋白質分解酵素（プロテアーゼＫやトリプシン等）を含むものが用いられている。この処理液は、蛋白質分解酵素で蛋白質を分解して、核酸を露出させ、プローブとのハイブリダイゼーション反応の機会を向上させる機能がある。しかしながら、蛋白質が分解されてしまうため、抗原の観測を行うことは不可能である。
このように従来使用されている前処理液を使用すると、細胞内の核酸と蛋白質を同時に観測することはできなかった。
また、ＤＮＡは酸に弱く、ＲＮＡはアルカリに弱いため、従来の前処理液ではＤＮＡとＲＮＡを同時に観測することは一般に極めて困難であった。
As a pretreatment solution for observing protein, a citrate buffer solution or a surfactant added thereto is used. The citrate buffer has a function of preventing the protein from being damaged during the heat treatment. In addition, the surfactant has a function of exposing the target protein in the cell and improving the chance of contact with the reactant supplied from the outside. However, the use of an autoclave is dangerous. In addition to the problem that the slide glass is broken and takes time, there is a problem that if the heat is excessively applied, the position of the protein in the cell is disturbed and the image information is destroyed.
In addition, as a pretreatment solution for observing nucleic acid, a solution containing a proteolytic enzyme (such as protease K or trypsin) is used. This treatment solution has a function of degrading a protein with a proteolytic enzyme to expose a nucleic acid and to improve the chance of a hybridization reaction with the probe. However, since the protein is degraded, it is impossible to observe the antigen.
As described above, when the conventionally used pretreatment solution is used, the intracellular nucleic acid and protein cannot be observed simultaneously.
In addition, since DNA is weak against acid and RNA is weak against alkali, it is generally very difficult to observe DNA and RNA simultaneously with a conventional pretreatment solution.
一方、染色体末端構造のひとつであるテロメアは、生体内における細胞分裂寿命を決定する重要な因子のひとつと考えられている。またテロメアは、精子や卵子を作る生殖細胞
以外の体細胞では細胞分裂のたびに短くなり、このテロメアの短縮が生体内における細胞分裂停止・細胞機能低下等の細胞老化を引き起こすと考えられている。同時に、細胞核内におけるテロメアの配置が細胞の分化・機能維持・機能低下に深く関与していることが示されている。従来の手法では細胞レベルでテロメアを観測することは可能であった（例えば、非特許文献１）。しかし、テロメアはＴＴＡＧＧＧの６塩基配列の繰り返し単位を有するため、ハイブリダイゼーションが難しく、このため、生体組織切片中のテロメアを蛍光in situハイブリダイゼーション法（ＦＩＳＨ）等の手法により観測し、生体組織切片の形態学的な情報とテロメアの動態を同時に得ることは不可能であった。
On the other hand, telomere, which is one of chromosome terminal structures, is considered to be one of the important factors that determine the cell division life in vivo. Telomeres are shortened at every cell division in somatic cells other than germ cells that produce sperm and eggs, and this shortening of telomeres is thought to cause cell aging such as cell division arrest and cell function decline in vivo. . At the same time, it has been shown that the arrangement of telomeres in the cell nucleus is deeply involved in cell differentiation / function maintenance / function decline. With conventional techniques, it was possible to observe telomeres at the cellular level (for example, Non-Patent Document 1). However, because telomeres have a repeating unit of 6 base sequences of TTAGGG, it is difficult to hybridize. For this reason, telomeres in biological tissue sections are observed by a technique such as fluorescence in situ hybridization (FISH). It was impossible to obtain morphological information and telomere dynamics simultaneously.
本発明の目的は、細胞内の核酸と蛋白質を同時に観測することができるような生体組織切片の前処理液を提供することである。
本発明の他の目的は、細胞内のＤＮＡとＲＮＡと蛋白質を同時に観測することができるような生体組織切片の前処理液を提供することである。
本発明のさらに他の目的は、テロメアＤＮＡとＲＮＡと蛋白質を同時に観測することができるような生体組織切片の前処理液を提供することである。
本発明のさらに他の目的は、上記前処理液を使用して、同一の生体組織切片の、核酸と蛋白質、ＤＮＡとＲＮＡと蛋白質、あるいはテロメアＤＮＡとＲＮＡと蛋白質の位置を同時に観測する方法を提供することである。
本発明のさらに他の目的は、上記前処理液を使用して、同一の生体組織切片の形態学的な情報とテロメアの動態を同時に観測する方法を提供することである。
An object of the present invention is to provide a pretreatment solution for a biological tissue section that can simultaneously observe intracellular nucleic acids and proteins.
Another object of the present invention is to provide a pretreatment solution for a biological tissue section so that intracellular DNA, RNA and protein can be observed simultaneously.
Still another object of the present invention is to provide a pretreatment solution for a biological tissue section so that telomeric DNA, RNA and protein can be observed simultaneously.
Still another object of the present invention is to provide a method for simultaneously observing the positions of nucleic acid and protein, DNA and RNA and protein, or telomeric DNA and RNA and protein in the same biological tissue section using the pretreatment solution. Is to provide.
Still another object of the present invention is to provide a method for simultaneously observing morphological information and telomere dynamics of the same biological tissue section using the pretreatment liquid.
本発明は以下の生体組織切片の前処理液及びこれを用いた生体組織切片中の核酸及び蛋白質の位置を観測する方法を提供するものである。
１．クエン酸緩衝液、キレート剤、及び下記式（Ｉ）で表されるポリエチレングリコールアルキルフェニルエーテルを含み、ｐＨが６．５〜７．５である、固定された生体組織切片の前処理液。
２．キレート剤がエチレンジアミン四酢酸塩である上記１記載の前処理液。
３．クエン酸ナトリウム、エチレンジアミン四酢酸ナトリウム、及び式（Ｉ）においてＲが８〜１０の直鎖及び／又は分岐鎖のアルキル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテルを含み、ｐＨが６．８〜７．０である、上記１記載の前処理液。
４．クエン酸ナトリウム濃度５〜２０ｍＭ、エチレンジアミン四酢酸ナトリウム濃度５〜２０ｍＭ、及び式（Ｉ）においてＲが８〜１０の直鎖及び／又は分岐鎖のアルキル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテル濃度０．０１〜０．２質量％、上記３記載の前処理液。
５．式（Ｉ）においてＲが直鎖及び／又は分岐鎖のオクチル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテルと式（Ｉ）においてＲが直鎖及び／又は分岐鎖のノニル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテルを含み、クエン酸ナトリウム濃度１０ｍＭ、エチレンジアミン四酢酸ナトリウム濃度１０ｍＭ、式（Ｉ）においてＲが直鎖及び／又は分岐鎖のオクチル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテルと式（Ｉ）においてＲが直鎖及び／又は分岐鎖のノニル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテルの合計の濃度０．１０質量％、ｐＨ７．０である、上記３記載の前処理液。
６．式（Ｉ）で表されるポリエチレングリコールアルキルフェニルエーテルが、式（Ｉ）においてＲが直鎖及び／又は分岐鎖のオクチル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテルと式（Ｉ）においてＲが直鎖及び／又は分岐鎖のノニル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテルの１：１混合物である、上記１〜５のいずれか１項記載の前処理液。
７．上記１〜６のいずれか１項記載の前処理液を使用することを特徴とする生体組織切片中の核酸及び蛋白質の同時観測方法。
８．核酸がＤＮＡ及びＲＮＡからなる群から選ばれる少なくとも１種である上記７記載の方法。
９．核酸がテロメアＤＮＡである上記８記載の方法。
１０．蛋白質が抗原蛋白質である上記８記載の方法。
１１．生体組織切片が凍結切片である上記７〜１０のいずれか１項記載の方法。
The present invention provides the following pretreatment liquid for biological tissue sections and a method for observing the positions of nucleic acids and proteins in biological tissue sections using the same.
1. A pretreatment solution for a fixed biological tissue section, which contains a citrate buffer, a chelating agent, and a polyethylene glycol alkylphenyl ether represented by the following formula (I) and has a pH of 6.5 to 7.5.
2. 2. The pretreatment liquid according to 1 above, wherein the chelating agent is ethylenediaminetetraacetate.
3. Including sodium citrate, sodium ethylenediaminetetraacetate, and polyethylene glycol alkylphenyl ether in which R is a linear and / or branched alkyl group of 8 to 10 in formula (I), and n is 8 to 10, 2. The pretreatment liquid according to 1 above, wherein is 6.8 to 7.0.
4). Polyethylene in which sodium citrate concentration is 5 to 20 mM, sodium ethylenediaminetetraacetate concentration is 5 to 20 mM, and in formula (I), R is a linear and / or branched alkyl group having 8 to 10 and n is 8 to 10 The pretreatment liquid according to 3 above, having a glycol alkylphenyl ether concentration of 0.01 to 0.2% by mass.
5. In formula (I), R is a linear and / or branched octyl group and n is 8 to 10, and in formula (I), R is a linear and / or branched nonyl group. A polyethylene glycol alkylphenyl ether in which n is 8 to 10, sodium citrate concentration 10 mM, ethylenediaminetetraacetate sodium concentration 10 mM, and in formula (I), R is a linear and / or branched octyl group , The total concentration of polyethylene glycol alkylphenyl ether in which n is 8 to 10 and polyethylene glycol alkylphenyl ether in which R is a linear and / or branched nonyl group in formula (I) and n is 8 to 10 4. The pretreatment liquid according to 3 above, which is 0.10% by mass and pH 7.0.
6). The polyethylene glycol alkylphenyl ether represented by the formula (I) is a polyethylene glycol alkylphenyl ether represented by the formula (I) wherein R is a linear and / or branched octyl group and n is 8 to 10. The former according to any one of 1 to 5 above, wherein in I) R is a linear and / or branched nonyl group and n is a 1: 1 mixture of polyethylene glycol alkylphenyl ethers of 8 to 10 Treatment liquid.
7). 7. A method for simultaneously observing nucleic acids and proteins in a biological tissue section, wherein the pretreatment liquid according to any one of 1 to 6 above is used.
8). 8. The method according to 7 above, wherein the nucleic acid is at least one selected from the group consisting of DNA and RNA.
9. 9. The method according to 8 above, wherein the nucleic acid is telomeric DNA.
10. 9. The method according to 8 above, wherein the protein is an antigen protein.
11. The method according to any one of 7 to 10 above, wherein the biological tissue section is a frozen section.
本発明の前処理液を使用すると、生体組織切片中の核酸及び蛋白質、ＤＮＡ及びＲＮＡ及び蛋白質の位置情報を同一の試料を用いて同時に観測することができる。また本発明の前処理液は、凍結された生体組織切片に対しても同様に適用することができる。
また本発明によれば従来観測が極めて困難であった生体組織切片での種々の分化過程にある細胞における核内のテロメアの動態を容易に観測することが可能である。
When the pretreatment liquid of the present invention is used, the position information of nucleic acids and proteins, DNA and RNA, and proteins in a biological tissue section can be simultaneously observed using the same sample. In addition, the pretreatment liquid of the present invention can be similarly applied to frozen biological tissue sections.
Further, according to the present invention, it is possible to easily observe the dynamics of telomeres in nuclei in cells in various differentiation processes in biological tissue sections that have been extremely difficult to observe conventionally.
本発明の前処理液は、生体組織を固定液で固定し、パラフィン包埋後に生体組織切片を作成した後、これを前処理して、生体組織切片中の標的物質を観測する際の当該前処理に使用される処理液である。
固定液としては、一般的に使用されるホルマリン系固定液、アルコール系固定液が使用できるが、標識済みプローブ等の組織切片への浸透性の点で、アルコール系固定液の方が好ましい。
本発明の前処理液は、クエン酸緩衝液とキレート剤、及び下記式（Ｉ）で表されるポリエチレングリコールアルキルフェニルエーテルを含み、ｐＨが６．５〜７．５である、固定された生体組織切片の前処理液である。
クエン酸緩衝液は熱処理時の蛋白質の損傷を防止し、蛋白質の細胞内の位置関係を維持する機能がある。クエン酸緩衝液としては、クエン酸ナトリウム緩衝液が一般的である。クエン酸緩衝液の濃度は１〜１００ｍＭ、好ましくは５〜２０ｍＭ、さらに好ましくは８〜１２ｍＭ、最も好ましくは１０ｍＭである。クエン酸緩衝液濃度が１ｍＭ未満では蛋白質の損傷を防止する効果が不充分となり、１００ｍＭを超えて使用する必要はない。
The pretreatment liquid of the present invention is prepared by fixing a biological tissue with a fixing solution, preparing a biological tissue section after embedding in paraffin, pretreating it, and observing the target substance in the biological tissue section. It is a processing liquid used for processing.
As the fixing solution, generally used formalin-based fixing solutions and alcohol-based fixing solutions can be used, but alcohol-based fixing solutions are preferred from the viewpoint of permeability to tissue sections such as labeled probes.
The pretreatment liquid of the present invention contains a citrate buffer, a chelating agent, and a polyethylene glycol alkylphenyl ether represented by the following formula (I), and has a pH of 6.5 to 7.5. It is a pretreatment liquid for tissue sections.
The citrate buffer has a function of preventing damage to the protein during heat treatment and maintaining the positional relationship of the protein in the cell. As the citrate buffer, a sodium citrate buffer is common. The concentration of the citrate buffer is 1-100 mM, preferably 5-20 mM, more preferably 8-12 mM, most preferably 10 mM. When the citrate buffer concentration is less than 1 mM, the effect of preventing protein damage is insufficient, and it is not necessary to use more than 100 mM.
キレート剤としては、エチレンジアミン四酢酸塩、プロピレンジアミン四酢酸等のナトリウム塩、カリウム塩等が挙げられるが、エチレンジアミン四酢酸塩ナトリウムが最も一般的である。キレート剤は、核酸分解酵素阻害剤として機能し、ＤＮＡやＲＮＡの分解を防止する。キレート剤の濃度は、試験環境中に存在する核酸の量によっても異なるが、通常は１〜１００ｍＭ、好ましくは５〜２０ｍＭ、さらに好ましくは８〜１２ｍＭ、最も好ましくは１０ｍＭである。キレート剤の濃度が１ｍＭより低いと核酸分解酵素阻害機能が不充分であり、１００ｍＭを超えて使用する必要はない。 Examples of the chelating agent include sodium salts such as ethylenediaminetetraacetic acid salt and propylenediaminetetraacetic acid, potassium salts and the like, and sodium ethylenediaminetetraacetate is the most common. The chelating agent functions as a nucleolytic enzyme inhibitor and prevents the degradation of DNA and RNA. The concentration of the chelating agent varies depending on the amount of nucleic acid present in the test environment, but is usually 1-100 mM, preferably 5-20 mM, more preferably 8-12 mM, most preferably 10 mM. If the concentration of the chelating agent is lower than 1 mM, the function of inhibiting nuclease is insufficient, and it is not necessary to use more than 100 mM.
本発明の前処理液は上記式（Ｉ）で表される特定のノニオン系界面活性剤を含有することを特徴とする。特に好ましい前処理液は、上記式（Ｉ）で表される特定のノニオン系界面活性剤を２種以上含むものである。好ましくは式（Ｉ）においてＲが直鎖及び／又は分岐鎖のオクチル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテルと式（Ｉ）においてＲが直鎖及び／又は分岐鎖のノニル基であり、ｎが８〜１０であるポリエチレングリコールアルキルフェニルエーテルの２：１〜１：２（質量比），好ましくは１：１混合物である。
式（Ｉ）においてＲが直鎖及び／又は分岐鎖のｐ−オクチル基であり、ｎが約８〜１０であるポリエチレングリコールアルキルフェニルエーテルの具体例としては、トリトンＸ−１００（ナカライテスク株式会社製商品名）が挙げられる。
他方のノニオン系界面活性剤である、式（Ｉ）においてＲが直鎖及び／又は分岐鎖のｐ−ノニル基であり、ｎが約８〜１０であるポリエチレングリコールアルキルフェニルエーテルの具体例としては、ＣＡＳ Ｎｏ．１２７０８７−８７−０（ＭＳＤＳ Ｎｏ．３２８２２）として登録され、タージトールＮＰ−４０（ナカライテスク株式会社製商品名）として市販されているものが挙げられる。
The pretreatment liquid of the present invention contains a specific nonionic surfactant represented by the above formula (I). A particularly preferred pretreatment liquid contains two or more kinds of specific nonionic surfactants represented by the above formula (I). Preferably, in formula (I), R is a linear and / or branched octyl group, n is 8 to 10 and polyethylene glycol alkylphenyl ether, and in formula (I), R is a linear and / or branched chain. It is a nonyl group and is a 2: 1 to 1: 2 (mass ratio), preferably 1: 1 mixture of polyethylene glycol alkylphenyl ethers in which n is 8 to 10.
Specific examples of the polyethylene glycol alkylphenyl ether in which R is a linear and / or branched p-octyl group and n is about 8 to 10 in the formula (I) include Triton X-100 (Nacalai Tesque Corporation) Product name).
Specific examples of the other nonionic surfactant, polyethylene glycol alkylphenyl ether in which R is a linear and / or branched p-nonyl group and n is about 8 to 10 in formula (I) CAS No. Examples include those registered as 127087-87-0 (MSDS No. 32822) and marketed as Taditol NP-40 (trade name, manufactured by Nacalai Tesque).
２種のノニオン系界面活性剤の濃度は、それぞれ好ましくは０．０１〜０．１質量％、さらに好ましくは０．０３〜０．０７質量％、最も好ましくは０．０５質量％である。
ノニオン系界面活性剤の濃度が０．１質量％より高いと、脆い臓器では溶解するおそれがあり、好ましくない。また、理由は不明であるが、２種の界面活性剤の一方が存在しないと核酸や蛋白質の形態及び位置関係が崩れてしまう場合があり、２種の界面活性剤を併用することが望ましい。
The concentrations of the two nonionic surfactants are each preferably 0.01 to 0.1% by mass, more preferably 0.03 to 0.07% by mass, and most preferably 0.05% by mass.
If the concentration of the nonionic surfactant is higher than 0.1% by mass, it may be dissolved in a brittle organ, which is not preferable. Although the reason is unknown, if one of the two surfactants does not exist, the form and positional relationship of the nucleic acid or protein may be disrupted, and it is desirable to use the two surfactants in combination.
本発明の前処理液のｐＨは６．５〜７．５、好ましくは６．７〜７．２、さらに好ましくは、６．８〜７．０、最も好ましくは７．０である。ｐＨが６．５より低いと酸に弱いＤＮＡの観測が困難になり、ｐＨが７．５より高いとアルカリに弱いＲＮＡの観測が困難になる。 The pH of the pretreatment liquid of the present invention is 6.5 to 7.5, preferably 6.7 to 7.2, more preferably 6.8 to 7.0, and most preferably 7.0. When the pH is lower than 6.5, it is difficult to observe DNA that is weak against acid, and when the pH is higher than 7.5, it is difficult to observe RNA that is weak against alkali.
本発明はさらに、上記前処理液を使用することを特徴とする生体組織切片中の核酸及び蛋白質の同時観測方法を提供するものである。
まず、生体組織を採取し、これを固定液により固定する。固定液としては、ホルマリン固定液や、メタカン固定液（メタノール：クロロホルム：酢酸＝６：３：１）等が使用できるが、後者の方が標識済みプローブ等の組織切片への浸透性の点で好ましい。
次に固定した組織をエタノール等で脱水後、常法に従いパラフィン包埋処理し、目的に応じた厚さ例えば、３〜１０μｍ程度の生体組織薄切切片を作成する。上記操作はいずれもＤＮａｓｅフリーのものを使用する。
The present invention further provides a method for simultaneously observing nucleic acids and proteins in a biological tissue section, characterized by using the pretreatment liquid.
First, a biological tissue is collected and fixed with a fixing solution. As the fixative, a formalin fixative or a metacan fixative (methanol: chloroform: acetic acid = 6: 3: 1) can be used, but the latter is more permeable to tissue sections such as labeled probes. preferable.
Next, after dehydrating the fixed tissue with ethanol or the like, paraffin embedding treatment is performed according to a conventional method, and a living tissue thin slice of about 3 to 10 μm is prepared according to the purpose. All of the above operations use DNase free.
脱パラフィン後、水洗し、本発明の前処理液中で所定時間、例えば、２０〜６０分間煮沸湯煎した後、水洗し、前処理液を除去する。
次に、切片に変性液、例えば、７０％ホルムアミド・２Ｘ ＳＳＣをのせ、加熱する。加熱温度、加熱時間、加熱手段は特に制限されないが、例えば、１００℃のヒートブロック上で５分間程度の加熱で充分である。
変性液を除去し、急冷脱水する。急冷脱水は例えば、ドライアイス入りのエタノールにより行うことができる。
After deparaffinization, it is washed with water, boiled hot water in a pretreatment liquid of the present invention for a predetermined time, for example, for 20 to 60 minutes, and then washed with water to remove the pretreatment liquid.
Next, a denaturing solution, for example, 70% formamide · 2X SSC is placed on the slice and heated. The heating temperature, heating time, and heating means are not particularly limited. For example, heating for about 5 minutes on a 100 ° C. heat block is sufficient.
Remove the denaturing solution and quench and dehydrate. The rapid cooling dehydration can be performed with ethanol containing dry ice, for example.
次いで常法により、標的物質と反応する物質を反応させるか、あるいは標的物質を着色可能な物質で着色する。核酸は、ＤＮＡ及びＲＮＡからなる群から選ばれる少なくとも１種であり、核酸の観測は、蛍光物質等で標識した核酸プローブをその場でハイブリダイズさせる（fluorescence in situ hybridization: FISH)。ハイブリダイゼーションは、例えば、蛍光標識した特異プローブを用いて、４５℃〜６０℃で１２〜２４時間、例えば、４５℃で一晩程度ハイブリダイズさせればよい。
次にクエン酸緩衝液例えば、０．１Ｘ ＳＳＣにより、４５℃で洗浄し、さらにリン酸緩衝液例えば、０．０１Ｍ−ＰＢＳで洗浄する。
次いで、常法により免疫組織化学・核染色を行う。
以上の処理を行った後、標識または着色された切片を蛍光顕微鏡や共焦点レーザー顕微鏡で観測することにより核酸及び蛋白質の位置を容易に観測することができる。
Next, a substance that reacts with the target substance is reacted or the target substance is colored with a colorable substance by a conventional method. The nucleic acid is at least one selected from the group consisting of DNA and RNA, and the nucleic acid is observed by hybridization of a nucleic acid probe labeled with a fluorescent substance or the like (fluorescence in situ hybridization: FISH). Hybridization may be performed, for example, by using a fluorescent-labeled specific probe at 45 ° C. to 60 ° C. for 12 to 24 hours, for example, at 45 ° C. overnight.
Next, it is washed with a citrate buffer solution, for example, 0.1X SSC at 45 ° C., and further washed with a phosphate buffer solution, for example, 0.01 M PBS.
Subsequently, immunohistochemistry and nuclear staining are performed by a conventional method.
After performing the above treatment, the position of the nucleic acid and protein can be easily observed by observing the labeled or colored section with a fluorescence microscope or a confocal laser microscope.
本発明は、ＤＮＡ、ＲＮＡ、蛋白質（例えば、抗原）を同時に観測できることを特徴とするものであるが、ＤＮＡ、ＲＮＡ、蛋白質（例えば、抗原）の１種の観測に適用できることはいうまでもない。
前処理液中の２種のノニオン系界面活性剤のうち一方のみを使用すると、生体組織切片中の核酸や蛋白質の形態（位置）が維持されなくなり、立体的な位置関係の情報が得られなくなることが多い。例えば、トリトンＸ−１００を含むがタージトールＮＰ−４０を含まない前処理液を使用すると、核酸に対するアクセス度が低下し、核酸の画像が消滅してしまう傾向がみられる。
The present invention is characterized in that DNA, RNA, and protein (for example, antigen) can be observed simultaneously. Needless to say, the present invention can be applied to one type of observation of DNA, RNA, and protein (for example, antigen). .
If only one of the two nonionic surfactants in the pretreatment liquid is used, the form (position) of the nucleic acid or protein in the biological tissue slice will not be maintained, and information on the three-dimensional positional relationship will not be obtained. There are many cases. For example, when a pretreatment liquid containing Triton X-100 but not containing Taditol NP-40 is used, the degree of access to the nucleic acid is lowered, and the nucleic acid image tends to disappear.
本発明は特定の緩衝液、キレート剤、特定の界面活性剤、好ましくは特に２種の特定の界面活性剤を組み合わせることにより、細胞内における核酸と蛋白質の立体的な位置情報を維持し、両者を同一の試料を用いて同一の視野内で観測することを可能としたものである。
本発明の方法は、生体、特に哺乳類の臓器を含む全ての組織の切片の観測に適用が可能である。また、凍結した切片を試料として用いることも可能である。
従来、核酸と蛋白質を同時に検出する手法は全く知られておらず、従って、本発明は核酸及び蛋白質の生体細胞内における画期的な動態観測手段を提案するものである。
The present invention maintains the three-dimensional positional information of nucleic acids and proteins in a cell by combining a specific buffer, a chelating agent, a specific surfactant, and preferably two types of specific surfactants. Can be observed within the same field of view using the same sample.
The method of the present invention can be applied to observation of sections of all tissues including living organisms, particularly mammalian organs. It is also possible to use a frozen section as a sample.
Conventionally, a method for simultaneously detecting a nucleic acid and a protein has not been known at all. Therefore, the present invention proposes an epoch-making means for observing the dynamic state of a nucleic acid and a protein in a living cell.
本発明の方法を適用して生体組織切片中のＤＮＡ、ＲＮＡ及び蛋白質（抗原等）を観測する方法においてその観測順序は特に制限されない。しかし、ＲＮＡが最も分解し易いのでＲＮＡを最初に観察することが通常は望ましい。次いで、蛋白質、ＤＮＡ、あるいはＤＮＡ、蛋白質の順に観測を行えば良い。 In the method of observing DNA, RNA and protein (antigen etc.) in a biological tissue slice by applying the method of the present invention, the observation order is not particularly limited. However, it is usually desirable to observe RNA first because it is most prone to degradation. Next, observation may be performed in the order of protein, DNA, or DNA, and protein.
実施例１
前処理液の調製
１０ｍＭクエン酸ナトリウム、１０ｍＭ ＥＤＴＡ、０．０５質量％タージトールＮＰ４０（ナカライテスク）及び０．０５質量％トリトンＸ−１００（ナカライテスク）を含む、ｐＨ７．０の前処理液を調製した。
Example 1
Preparation of Pretreatment Solution A pH 7.0 pretreatment solution containing 10 mM sodium citrate, 10 mM EDTA, 0.05 mass% Taditol NP40 (Nacalai Tesque) and 0.05 mass% Triton X-100 (Nacalai Tesque) was prepared. did.
試験例１
マウス精巣を摘出し、メタカン固定液（メタノール：クロロホルム：酢酸＝６：３：１）により固定した。使用した器具はＤＮａｓｅフリーのものを使用した。
固定した組織をエタノール脱水後、常法に従いパラフィン包埋した。使用した器具はＤＮａｓｅフリーのものを使用した。
ミクロトームを用いて厚さ５μｍの生体組織切片を作成した。使用した器具はＤＮａｓｅフリーのものを使用した。
脱パラフィン後、水洗し、実施例１で調製した前処理液中で４０分間煮沸湯煎した。
水洗し、前処理液を除去した。
切片に変性液（７０％ホルムアミド・２Ｘ ＳＳＣ）をのせ、１００℃のヒートブロック上で５分間加熱した。
変性液を除去し、ドライアイス入りのエタノールで急冷脱水した。
０．２μｇ／ｍｌローダミンで蛍光標識したテロメア特異プローブ（ＴＴＡＧＧＧ）3を用いて、５０℃１２時間ハイブリダイズした。
０．０１Ｘ ＳＳＣ、４５℃で洗浄した。
０．０１Ｍ−ＰＢＳで洗浄した。
Test example 1
The mouse testis was excised and fixed with a metacan fixing solution (methanol: chloroform: acetic acid = 6: 3: 1). The instrument used was DNase free.
The fixed tissue was dehydrated with ethanol and embedded in paraffin according to a conventional method. The instrument used was DNase free.
A biological tissue section having a thickness of 5 μm was prepared using a microtome. The instrument used was DNase free.
After deparaffinization, it was washed with water and boiled for 40 minutes in the pretreatment liquid prepared in Example 1.
Washed with water to remove the pretreatment liquid.
Denatured solution (70% formamide · 2X SSC) was placed on the sections and heated on a heat block at 100 ° C. for 5 minutes.
The denatured solution was removed, and rapid dehydration was performed with ethanol containing dry ice.
Hybridization was carried out at 50 ° C. for 12 hours using a telomere-specific probe (TTAGGG) 3 fluorescently labeled with 0.2 μg / ml rhodamine.
Washed at 0.01X SSC, 45 ° C.
Washed with 0.01M PBS.
次に上記試料をサイトグリーン（Molecular Probes, USA）により核染色（緑色）を行い、さらに抗ＰＣＮＡモノクローナル抗体（Oncogene Research Products, USA)及びAlexa Flour（登録商標）６３３で標識した第２抗体（Molecular Probes, USA）と反応させ免疫組織染色（青色）を行った。
蛍光顕微鏡を用いてマウス精巣におけるテロメア動態を観察し、ＦＩＳＨ画像（赤色）を得た。
また、ＬＳＭ−５１０共焦点顕微鏡（Carl Zeiss, Germany)により、核染色した画像（緑色）及び免疫組織染色した画像（青色）を得た。
３種の画像（免疫組織染色画像（青色）／ＦＩＳＨ画像（赤色）／核染色画像（緑色））を合成した画像を作成したところ、３色の画像に加え、赤色と緑色が合成されて黄色となった部分、緑色と青色が合成されて水色となった部分、青色と赤色が合成されて赤紫色となった部分、これらの中間色の部分が観測された。この試験では全く同一の試料を用いており、これにより、生体組織切片中のテロメアＤＮＡと特定の抗原及び核の位置関係を明瞭に確認することができた。
Next, the sample was subjected to nuclear staining (green) with Cytogreen (Molecular Probes, USA), and a second antibody (Molecular) labeled with anti-PCNA monoclonal antibody (Oncogene Research Products, USA) and Alexa Flour (registered trademark) 633. Probes, USA) and immunohistochemical staining (blue).
The telomere dynamics in the mouse testis were observed using a fluorescence microscope, and a FISH image (red) was obtained.
In addition, images with nuclear staining (green) and images with immune tissue staining (blue) were obtained using an LSM-510 confocal microscope (Carl Zeiss, Germany).
An image was created by synthesizing three types of images (immunological tissue staining image (blue) / FISH image (red) / nuclear staining image (green)). In addition to the three colors, red and green were synthesized and yellow. The part that became green, the part that became green with the combination of green and blue, the part that became blue and red with the combination of blue and red, and the part of the intermediate color were observed. In this test, exactly the same sample was used, and thereby, the positional relationship between the telomeric DNA, the specific antigen and the nucleus in the biological tissue section could be clearly confirmed.
試験例２（比較）
試験例１において、実施例１の前処理液の代わりに、１質量％トリトン含有ＰＢＳを使用したもの、および前処理液を使用しなかったものでは、テロメアのＦＩＳＨ画像は極めて不明瞭であり、テロメアと、特定の抗原及び核の位置関係を明瞭に確認することはできなかった。
Test example 2 (comparison)
In Test Example 1, in place of the pretreatment liquid of Example 1 in which PBS containing 1% by weight of triton was used and in the case where no pretreatment liquid was used, the telomere FISH image was very unclear, The positional relationship between telomeres, specific antigens, and nuclei could not be clearly confirmed.
試験例１において、マウスの精巣の代わりにマウス胎子脳を使用し、Ｐａｘ６のｍＲＮＡとその遺伝子産物の同定を同時に行った。Ｐａｘ６・ｍＲＮＡの存在を示すシグナルはディゴキシゲニン標識した特異プローブを用いたin situ ハイブリダイゼーションによって試行し（疑似カラー・緑色で表示）、Ｐａｘ６・遺伝子産物（この場合は蛋白質）の存在はＰａｘ６に対する特異抗体を用いた免疫組織化学にて試行した（疑似カラー・赤色で表示）。なお、対比核染色としてはＤＮＡ染色剤を用いた（疑似カラー・青色で表示）。 In Test Example 1, mouse fetal brain was used instead of the mouse testis, and Pax6 mRNA and its gene product were simultaneously identified. The signal indicating the presence of Pax6 · mRNA was tested by in situ hybridization using a digoxigenin-labeled specific probe (pseudo-color, green), and the presence of Pax6 · gene product (in this case protein) is a specific antibody against Pax6. Trial was carried out by immunohistochemistry using a ply (displayed in pseudo color and red). In addition, a DNA stain was used as the counter-nuclear staining (displayed in pseudo color and blue).
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| JP2004525366A (en) * | 2001-03-07 | 2004-08-19 | ベンタナ・メディカル・システムズ・インコーポレーテッド | Automated immunohistochemistry and in situ hybridization assay formulations |
| WO2004086945A2 (en) * | 2003-03-31 | 2004-10-14 | Cytyc Corporation | Papanicolau staining process |
| JP2007532918A (en) * | 2004-04-16 | 2007-11-15 | チュー，ウェイシン | Biomolecule extraction device from tissue specimens |
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| JP2004525366A (en) * | 2001-03-07 | 2004-08-19 | ベンタナ・メディカル・システムズ・インコーポレーテッド | Automated immunohistochemistry and in situ hybridization assay formulations |
| WO2004086945A2 (en) * | 2003-03-31 | 2004-10-14 | Cytyc Corporation | Papanicolau staining process |
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