JP2002031586A - Bio-tissue freezing slicing method, slice preservation method and bio-tissue freezing slicing device - Google Patents
Bio-tissue freezing slicing method, slice preservation method and bio-tissue freezing slicing deviceInfo
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- JP2002031586A JP2002031586A JP2000393801A JP2000393801A JP2002031586A JP 2002031586 A JP2002031586 A JP 2002031586A JP 2000393801 A JP2000393801 A JP 2000393801A JP 2000393801 A JP2000393801 A JP 2000393801A JP 2002031586 A JP2002031586 A JP 2002031586A
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- frozen
- sample
- plastic film
- adhesive
- slicing
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、硬組織（例えば、
骨や歯等）や大きな生物試料（例えば、実験動物の全
身）から生命科学の研究（例えば、組織学的研究、組織
化学的研究、酵素組織化学的研究、免疫組織化学的研
究、遺伝子組織化学的研究、水溶性物質の体内分布研
究）に利用できる薄い凍結切片（厚さが２〜１０μｍ）
を得るのに好適な生物組織凍結薄切方法、切片を長期保
存するための切片保存方法及び生物組織凍結薄切装置に
関するものである。The present invention relates to a hard tissue (for example,
Life science studies (eg, histological studies, histochemical studies, enzyme histochemical studies, immunohistochemical studies, gene histochemistry) from large biological samples (eg, whole body of experimental animals) Frozen slices (2 to 10 μm in thickness) that can be used for scientific research and biodistribution studies of water-soluble substances)
The present invention relates to a method for cryosection of a biological tissue, a method for storing a slice for long-term preservation of a slice, and a device for cryosection of a biological tissue suitable for obtaining a tissue.
【０００２】[0002]
【従来の技術】生命科学の研究では、化学薬品により固
定処理、脱灰処理されていない（以後、未固定非脱灰と
呼ぶ）硬組織や大きな生物試料から作製される薄い凍結
切片（厚さ２〜１０μｍ）が必要とされている。しか
し、従来の生物組織凍結薄切方法と生物組織凍結薄切装
置では、小さな軟組織（例えば、薄切面が２ｃｍ×２ｃ
ｍの大きさ）から厚さ５μｍ程度の凍結切片を作製する
のが限界で、組織の形態が良好に保たれた大きな凍結切
片、あるいは更に薄い凍結切片（例えば、厚さが２μ
ｍ）を作製することは極めて困難であった。また、硬組
織から凍結切片を作製することはできなかった。従っ
て、そのような切片を封入保存するのに適した簡便な方
法がない。2. Description of the Related Art In life science research, thin frozen sections (thicknesses) made from hard tissues or large biological samples that have not been fixed or decalcified (hereinafter referred to as unfixed non-decalcified) with chemicals have been used. 2 to 10 μm) are required. However, in the conventional biological tissue cryosection method and the biological tissue cryosection device, a small soft tissue (for example, a section of 2 cm × 2 c
It is limited to produce a frozen section with a thickness of about 5 μm from a large frozen section with a good tissue morphology or a thinner frozen section (eg, 2 μm thick).
m) was extremely difficult to produce. In addition, frozen sections could not be prepared from hard tissues. Therefore, there is no simple method suitable for enclosing and storing such sections.
【０００３】[0003]
【発明が解決しようとする課題】従来の生物組織凍結薄
切方法、切片保存方法、凍結包埋容器、生物組織凍結薄
切装置には、それぞれ下記に示す問題があり、未固定非
脱灰の硬組織や大きな生物試料（例えば、薄切面が４ｃ
ｍ×５ｃｍの大きさ）から光学顕微鏡観察に利用できる
薄い凍結切片（例えば、厚さが２μｍ）を作製し、長期
保存することができなかった。The conventional methods for cryosectioning biological tissues, section preserving methods, cryo-embedding containers, and cryosectioning apparatus for biological tissues have the following problems, respectively. Hard tissue or large biological samples (eg, 4c thin section)
A thin frozen section (for example, having a thickness of 2 μm) that can be used for optical microscopic observation was prepared from a sample having a size of mx 5 cm) and could not be stored for a long time.
【０００４】生物組織凍結薄切方法の問題点 従来の生物組織凍結薄切方法では、体内物質の分布状態
に影響を与えないで、凍結薄切を実施する環境下（例え
ば、−２５℃の冷凍庫内）で凍結切片を支持することが
できなかった。そのため、凍結薄切の実施中に凍結切片
が簡単に壊れ、硬組織や大きな生物試料から組織の形態
が良好に保たれた薄い凍結切片を作製することができな
い欠点がある。Problems of Freezing and Slicing of Biological Tissue In the conventional method of freezing and slicing biological tissue, the freezing and slicing method is performed in an environment where freezing and slicing is performed without affecting the distribution state of substances in the body (for example, a freezer at -25 ° C. In), frozen sections could not be supported. For this reason, there is a disadvantage that the frozen section is easily broken during the freeze-thinning, and it is not possible to produce a thin frozen section in which the morphology of the tissue is well maintained from a hard tissue or a large biological sample.
【０００５】切片保存方法の問題点 従来の方法で作製される小さな凍結切片（例えば、薄切
面が２ｃｍ×２ｃｍの大きさ）は、透明なガラス板（例
えば、厚さが１ｍｍのガラス板）と薄いガラス板（例え
ば、厚さが０．１ｍｍのガラス板）の間に封入剤を用い
て長期保存できる。しかし、本方法で作製される粘着剤
を塗布したプラスチックフィルムに貼り付いた大きな切
片は、プラスチックフィルムが封入の妨げとなり、従来
の方法で封入することが困難である。Problems of section storage method [0005] A small frozen section (for example, a thin section having a size of 2 cm x 2 cm) produced by a conventional method is made of a transparent glass plate (for example, a glass plate having a thickness of 1 mm). It can be stored for a long time between thin glass plates (for example, a glass plate having a thickness of 0.1 mm) using an encapsulating agent. However, a large piece attached to a plastic film coated with an adhesive produced by the present method is difficult to enclose by a conventional method because the plastic film hinders encapsulation.
【０００６】凍結包埋容器の問題点 従来の方法では、試料の急速凍結を必要としないために
水溶性包埋剤の急速凍結に適した包埋容器が製作されて
いない。そのため、水溶性包埋剤中に入れた凍結試料が
水溶性包埋剤の凍結中に溶解する欠点がある。Problems of the frozen embedding container [0006] In the conventional method, since the rapid freezing of the sample is not required, an embedding container suitable for the quick freezing of the water-soluble embedding agent has not been manufactured. Therefore, there is a drawback that a frozen sample placed in a water-soluble embedding medium dissolves during freezing of the water-soluble embedding medium.
【０００７】生物組織凍結薄切装置の問題点 現在、凍結した未固定非脱灰の硬組織や大きな生物試料
を切削できる装置として、全身オートラジオグラフィー
に用いる厚い切片（例えば、厚さが５０μｍ）を作製す
るための装置（以後、大型凍結薄切装置と呼ぶ）があ
る。この大型凍結薄切装置で光学顕微鏡観察に利用でき
る薄い凍結切片（例えば、厚さが２μｍ）を作製するに
は、次の欠点がある。 ・硬組織から組織構造が良好に保たれた薄い凍結切片を
安定して作製する刃物としては、硬組織を薄切できるタ
ングステンカーバイド製の替刃が最適であるが、従来の
大型凍結薄切装置には、その替刃を固定する装置がな
く、替刃を用いることができない。 ・従来の大型凍結薄切装置に取り付けられている試料傾
斜装置は、傾斜範囲が小さく、目的の組織構造を正確に
露出させることができない。 ・従来の大型凍結薄切装置で薄い凍結切片（例えば、厚
さが２μｍ）を作製するには、大型凍結薄切装置を設置
している冷凍庫の扉を全開して作業をしなければならな
い。そのため、作業中に冷凍庫内の温度が上昇し、組織
構造の良く保存された凍結切片の作製に最適な温度を維
持することができない。Problems with the biological tissue cryosectioning device At present, as a device capable of cutting frozen, unfixed, undecalcified hard tissue or a large biological sample, a thick section (for example, 50 μm in thickness) used for whole body autoradiography. (Hereinafter, referred to as a large freezing slicer). The production of thin frozen sections (for example, 2 μm thick) that can be used for optical microscopic observation with this large-sized frozen sectioning apparatus has the following disadvantages. -A tungsten carbide spare blade that can slice hard tissue is the best tool for stably producing thin frozen sections with good tissue structure maintained from hard tissue. Does not have a device for fixing the replacement blade, and cannot use the replacement blade. -The sample inclining device attached to the conventional large-sized cryosectioning device has a small inclining range and cannot accurately expose a target tissue structure. In order to produce thin frozen sections (for example, 2 μm in thickness) using a conventional large-sized freeze-thickening device, the work must be performed by fully opening the door of a freezer in which the large-size freeze-thickness-slicing device is installed. For this reason, the temperature in the freezer rises during the operation, and it is not possible to maintain the optimal temperature for producing a frozen section having a well-preserved tissue structure.
【０００８】上記の問題があるために、従来の生物組織
凍結薄切方法と大型凍結薄切装置では、凍結した硬組織
や大きな生物試料から、生命科学の研究に利用できる薄
い凍結切片（例えば、厚さが２μｍ）を作製することが
できなかった。[0008] Because of the above-mentioned problems, conventional methods for cryosectioning biological tissues and large-sized cryosectioning apparatuses use thin frozen sections (eg, for example) from frozen hard tissues or large biological samples that can be used for life science research. (Thickness: 2 μm) could not be produced.
【０００９】本発明は、上記の問題点を解決するため
に、低温下（例えば、−２５℃）でも凍結切片を粘着支
持でき、生命科学の研究に必要な処理（例えば、水やア
ルコールを用いた処理）に耐えることができる粘着剤と
プラスチックフィルム、凍結薄切に適した形状の凍結試
料を作製するための容器、硬組織や大きな生物試料から
薄い凍結切片（例えば、厚さが２μｍ）を作製すること
ができる凍結薄切装置を用いて、硬組織や大きな生物試
料から生命化学の研究に利用できる薄い凍結切片を効率
良く作製し、その切片を長期保存しようとするものであ
る。In order to solve the above problems, the present invention is capable of adhesively supporting a frozen section even at a low temperature (for example, -25 ° C.) and using a treatment (for example, using water or alcohol) required for research in life science. Pressure-sensitive adhesive and plastic film that can withstand cold processing, containers for preparing frozen samples in a shape suitable for freeze-thin sectioning, thin frozen sections (eg, 2 μm thick) from hard tissues and large biological samples. Using a cryosectioning device that can be prepared, thin cryosections that can be used for biochemical research can be efficiently prepared from hard tissues or large biological samples, and the sections are to be stored for a long period of time.
【００１０】[0010]
【課題を解決するための手段】本発明は、水溶性包埋剤
中に凍結包埋した生物試料（例えば、未固定非脱灰の硬
組織、実験動物の全身）を凍結薄切装置に固定し、その
固定した試料の所定面に粘着剤を塗布した薄いプラスチ
ックフィルム（例えば、厚さが１０μｍのポリ塩化ビニ
リデンフィルム）（以後、粘着プラスチックフィルムと
呼ぶ）を貼り付け、粘着プラスチックフィルムが貼り付
いた試料を、所定の刃物により粘着プラスチックフィル
ムが貼り付いた状態で、所定の厚さに薄切しようとする
ものである。SUMMARY OF THE INVENTION According to the present invention, a biological sample (eg, unfixed, non-decalcified hard tissue, whole body of an experimental animal) frozen embedded in a water-soluble embedding medium is fixed to a cryosectioning apparatus. Then, a thin plastic film (for example, a polyvinylidene chloride film having a thickness of 10 μm) coated with an adhesive (hereinafter referred to as an adhesive plastic film) is attached to a predetermined surface of the fixed sample, and the adhesive plastic film is attached. The sample is to be sliced to a predetermined thickness in a state where the adhesive plastic film is adhered by a predetermined blade.
【００１１】[0011]
【発明の実施の形態】以下、添付図面を参照して、本発
明の一実施の形態について説明する。An embodiment of the present invention will be described below with reference to the accompanying drawings.
【００１２】本例においては、水溶性包埋剤（例えば、
５％カルボキシメチルセルロース）中に凍結包埋した生
物試料を薄切するようにしたものである。まず、水溶性
包埋剤中に生物試料を凍結包埋させる容器について、図
１と図２を参照して説明する。図１に示す凍結包埋容器
の容器部２は、凍結した水溶性包埋剤の取り出しを容易
にするために、次のような構造となっている。In this example, a water-soluble embedding agent (for example,
Biological samples frozen embedded in 5% carboxymethylcellulose) were sliced. First, a container for freezing and embedding a biological sample in a water-soluble embedding medium will be described with reference to FIGS. The container portion 2 of the frozen embedding container shown in FIG. 1 has the following structure in order to facilitate removal of the frozen water-soluble embedding agent.
【００１３】図１に示すように、凍結包埋容器の容器部
２は箱型で、金属板（例えば、厚さが１．０ｍｍのステ
ンレス板）で作られ、凍結作業を迅速に行うために取っ
手１を容器部２の側面に付けている。この容器部２内で
凍結した水溶性包埋剤の凍結包埋ブロック５を取り出し
やすいように、容器部２の上面が底面より大きくなって
いる。容器部２の上面は、凍結ブロックを取り出すため
に取り除いている。底面は下蓋３として、図２に示すよ
うに容器側面に付けた下蓋固定用Ｌ型金具４で固定さ
れ、引き抜くことができる。As shown in FIG. 1, the container portion 2 of the frozen embedding container is box-shaped and made of a metal plate (for example, a stainless steel plate having a thickness of 1.0 mm). The handle 1 is attached to the side of the container 2. The upper surface of the container 2 is larger than the bottom so that the frozen embedding block 5 of the water-soluble embedding agent frozen in the container 2 can be easily taken out. The upper surface of the container part 2 is removed to take out the frozen block. The bottom surface is fixed as a lower lid 3 by a lower lid fixing L-shaped bracket 4 attached to the side of the container as shown in FIG. 2, and can be pulled out.
【００１４】本例においては、凍結包埋容器の容器部２
から取り出した凍結包埋ブロック５（図２参照）は、上
記の生命科学の研究に利用するために、厚さ２〜１０μ
ｍに薄切される。この薄切を実施するために、図３に示
すように、凍結包埋ブロック５を金属製の試料台６に固
定する。この試料台６は、金属板（例えば、厚さが２．
０ｍｍのステンレス板）で作られている。試料台６に
は、凍結包埋ブロック固定用突起７（図３のＡ参照）を
複数個設け、図３のＢに示すように試料台６に凍結包埋
ブロック５を固定する。この試料台６は、後述する図９
に示すように、試料台固定ネジ２８で試料傾斜装置８に
固定される。In this embodiment, the container portion 2 of the frozen embedding container is used.
The frozen embedding block 5 (see FIG. 2) taken out of the above is used for the above-mentioned life science research, and has a thickness of 2 to 10 μm.
m. In order to perform this slicing, the frozen embedding block 5 is fixed to a metal sample stand 6 as shown in FIG. The sample table 6 is made of a metal plate (for example, having a thickness of 2.
0 mm stainless steel plate). A plurality of projections 7 (see FIG. 3A) for fixing a frozen embedded block are provided on the sample table 6, and the frozen embedded block 5 is fixed to the sample table 6 as shown in FIG. 3B. This sample stage 6 is shown in FIG.
As shown in (1), it is fixed to the sample tilting device 8 by the sample stage fixing screw 28.
【００１５】試料台６に固定した凍結包埋ブロック５
は、図４〜図６に示すタングステンカーバイド製替刃用
支持具１１（以後、替刃支持具と呼ぶ）に固定したタン
グステンカーバイド製替刃９（以後、替刃と呼ぶ）で薄
切される。替刃支持具１１は、金属（例えば、特殊鋼）
で作られ、替刃９を固定する。替刃支持具１１の替刃固
定部１７は、図５のＢの断面図に示すように替刃９の刃
先と一体となるような形状となっている。図５のＡは、
替刃９、替刃押さえ金具１０、替刃支持具１１を分解し
て示す断面図であり、図５のＢは組み立てた状態を示す
断面図である。A frozen embedding block 5 fixed to a sample table 6
Is sliced by a tungsten carbide spare blade 9 (hereinafter, referred to as a spare blade) fixed to the tungsten carbide spare blade support 11 (hereinafter, referred to as a spare blade support) shown in FIGS. . The blade support 11 is made of metal (for example, special steel)
And fix the spare blade 9. The replacement blade fixing portion 17 of the replacement blade support 11 has a shape that is integrated with the cutting edge of the replacement blade 9 as shown in the cross-sectional view of FIG. A of FIG.
FIG. 5 is an exploded cross-sectional view of the replacement blade 9, the replacement blade holding member 10, and the replacement blade support 11, and FIG. 5B is a cross-sectional view illustrating an assembled state.
【００１６】替刃９の装着では、刃先の向きが特に重要
である。組織の形態が良好に保たれた切片を作製するた
めに、図５に示すように切削角が最小となるように取り
付ける。替刃押さえ金具固定ネジ１６を緩めることによ
り、凍結包埋ブロック５の薄切面との位置関係を保った
状態で新しい替刃に交換できる。In mounting the replacement blade 9, the direction of the blade edge is particularly important. In order to prepare a section in which the morphology of the tissue is well maintained, the section is mounted so as to minimize the cutting angle as shown in FIG. By loosening the blade fixing screw 16, the blade can be replaced with a new blade while maintaining the positional relationship with the thin section of the frozen embedding block 5.
【００１７】替刃支持具固定台１２は、図６と図７に示
すように、替刃支持具１１を保持する。この替刃支持具
固定台１２は、金属（例えば、ステンレス）で作られて
いる。替刃支持具固定台１２への替刃支持具１１の固定
は、替刃支持具固定ネジ１８で行う。替刃支持具固定台
１２の左右の端部には、図６と図７に示すように、替刃
支持具固定台１２を凍結薄切装置に固定する替刃支持具
固定台の取付け金具１３に替刃支持具固定台固定ネジ１
９で固定し、この取付け金具１３を介して、図４に示す
ように凍結薄切装置に固定する。The replacement blade support fixture 12 holds the replacement blade support 11 as shown in FIGS. The replacement blade support fixture base 12 is made of metal (for example, stainless steel). The replacement blade support 11 is fixed to the replacement blade support fixing base 12 with a replacement blade support fixing screw 18. As shown in FIGS. 6 and 7, mounting brackets 13 of the blade support fixture fixing table for fixing the blade support fixture 12 to the freezing and slicing device are provided on the left and right ends of the blade support fixture 12. Blade fixing tool fixing stand fixing screw 1
9, and is fixed to the freeze-thinning device via the mounting bracket 13 as shown in FIG.
【００１８】次に、本例の試料である凍結包埋ブロック
５を傾斜させる試料傾斜装置８について、図４、図８，
図９を参照して説明する。試料傾斜装置８は、金属（例
えば、ステンレス）で作られ、直交する２つの軸である
ｘ軸とｙ軸に独立して傾斜可能としてある。具体的に
は、ｙ方向回転軸２０は、ｙ方向回転軸固定金具２１に
よりｘ方向回転軸２３に直交して固定されている。試料
台６は、試料台傾斜固定ネジ２７を緩めることにより、
ｙ方向回転軸２０を中心に±１５度以上傾斜できる。ま
た、ｘ方向回転軸固定金具固定ネジ２６を緩めることに
より、Ｘ方向回転軸２３を中心に試料台を±１５度以上
傾斜できる。図８には、試料傾斜装置８の構成部品を示
し、図４と図９には、試料台６、試料傾斜装置８、削り
かす容器１４を組み立てた斜視図を示している。Next, a sample tilting device 8 for tilting the frozen embedding block 5, which is a sample of the present example, will be described with reference to FIGS.
This will be described with reference to FIG. The sample tilting device 8 is made of metal (for example, stainless steel), and can be tilted independently of two orthogonal axes, x-axis and y-axis. Specifically, the y-direction rotation shaft 20 is fixed orthogonal to the x-direction rotation shaft 23 by a y-direction rotation shaft fixing bracket 21. By loosening the sample stage inclination fixing screw 27, the sample stage 6
It can be tilted ± 15 degrees or more around the y-axis rotation axis 20. In addition, by loosening the fixing screw 26 for fixing the x-axis rotation axis fixing bracket, the sample stage can be inclined ± 15 degrees or more about the X-axis rotation axis 23. FIG. 8 shows the components of the sample tilting device 8, and FIGS. 4 and 9 show perspective views in which the sample stage 6, the sample tilting device 8, and the shaving container 14 are assembled.
【００１９】本例の試料傾斜装置８は、金属板（例え
ば、厚さが２．０ｍｍのステンレス板）で作られている
削りかす容器１４の底板に固定されている。削りかす容
器１４の底板には、削りかすを廃棄する廃棄口１５を設
けてある。この削りかす容器１４は、大型凍結薄切装置
の試料台２５にネジで固定できる構造となっている。The sample tilting device 8 of this embodiment is fixed to a bottom plate of a shaving vessel 14 made of a metal plate (for example, a stainless plate having a thickness of 2.0 mm). The bottom plate of the shavings container 14 is provided with a disposal port 15 for discarding shavings. The shavings container 14 has a structure that can be fixed to a sample table 25 of a large-sized freezing and slicing apparatus with screws.
【００２０】凍結薄切装置を設置する冷凍庫の上蓋は、
図１０と図１１に示すように、２重の蓋となるような構
成としている。透明なプラスチック製（例えば、アクリ
ル）固定板３１と引戸３０を、従来の開き戸式開閉蓋２
９の直下に取り付けている。作業口の大きさは、引戸３
０を開閉することにより調節できる。また、冷気循環用
送風機３２を冷凍庫の底部に設置し、矢印３３の方向に
冷気を循環させるようにしている。The upper lid of the freezer in which the freeze slicing device is installed is:
As shown in FIG. 10 and FIG. 11, it is configured to be a double lid. A transparent plastic (for example, acrylic) fixing plate 31 and a sliding door 30 are connected to a conventional hinged door 2
It is installed just below 9. The size of the working port is sliding door 3.
It can be adjusted by opening and closing 0. In addition, a cool air circulation blower 32 is installed at the bottom of the freezer to circulate cool air in the direction of arrow 33.
【００２１】次に、凍結試料を薄切する際に、凍結切片
の支持材として用いる粘着プラスチックフィルムの作製
手順について、図１２と図１３を参照して説明する。ま
ず、図１３に示すように、化学薬品に強く、透明で、柔
軟性があり、しかも薄くて伸縮性の小さいプラスチック
フィルム３５（例えば、厚さが１０μｍのポリ塩化ビニ
リデン製フィルム）を、アルコールを塗布した透明なア
クリル板３９に密着する。次いで、凍結包埋ブロック５
の薄切面の大きさの粘着プラスチックフィルムを作製す
るために、図１２に示すように粘着剤３４（例えば、−
２５℃で粘着力を保持しているゴム系粘着剤、あるいは
アクリル系粘着剤）を薄切面の長辺の幅と同じ幅（粘着
剤塗布幅３８）に塗布する。この粘着剤の塗布を実施す
る時、図１２と図１３に示すように、プラスチックフィ
ルム上に粘着剤を塗布しない領域（粘着剤未塗布域３
７）を残す。粘着剤３４の乾燥後、粘着プラスチックフ
ィルムを薄切面の短辺の幅に合わせて回転式カッターを
用いて切断する。図１２に、その切断個所３６を示して
いる。Next, a procedure for preparing an adhesive plastic film used as a support for a frozen section when the frozen sample is sliced will be described with reference to FIGS. First, as shown in FIG. 13, a plastic film 35 (for example, a polyvinylidene chloride film having a thickness of 10 μm) that is strong against chemicals, transparent, flexible, thin, and has a small elasticity is treated with alcohol. The transparent acrylic plate 39 is adhered to the applied transparent acrylic plate 39. Next, the frozen embedding block 5
As shown in FIG. 12, an adhesive 34 (for example, −
A rubber pressure-sensitive adhesive or an acrylic pressure-sensitive adhesive having an adhesive force at 25 ° C. is applied to the same width as the width of the long side of the thin section (adhesive application width 38). When this adhesive is applied, as shown in FIGS. 12 and 13, the area where the adhesive is not applied (the area where the adhesive is not applied 3
7) Leave. After the adhesive 34 is dried, the adhesive plastic film is cut with a rotary cutter in accordance with the width of the short side of the thin section. FIG. 12 shows the cut portion 36.
【００２２】次に、上記で説明した替刃支持具１１と試
料傾斜装置８を取り付けた大型凍結薄切装置と粘着プラ
スチックフィルムを用いて、生物試料から凍結切片を作
製する生物組織凍結薄切方法について順に説明する。Next, a biological tissue freeze-slicing method for preparing a frozen slice from a biological sample using the large-sized freeze-slicing device equipped with the replacement blade support 11 and the sample tilting device 8 described above and an adhesive plastic film. Will be described in order.
【００２３】ｉ）凍結試料と水溶性包埋剤を、図１に示
す凍結包埋容器の容器部２に入れる。 ｉｉ）凍結包埋容器の取っ手１を持って、容器部２を冷
媒中（例えば、−９４℃のヘキサン）に入れて凍結す
る。 ｉｉｉ）凍結後、図２に示すように、凍結包埋容器の容
器部２の下蓋３を抜き取り、凍結包埋ブロック５を下か
ら押し出す。 ｉｖ）取り出した凍結包埋ブロック５を、図３に示すよ
うに試料台６に固定し、図４、図８，図９に示す試料傾
斜装置８に試料台固定ネジ２８で固定する。 ｖ）上記の手順で作製した粘着プラスチックフィルム
を、図１４に示すように凍結包埋ブロック５の薄切面に
貼り付ける。 ｖｉ）この状態で、替刃支持具１１に固定した替刃９で
薄切を実施することにより、凍結切片４０が粘着プラス
チックフィルムに貼り付いた状態で採れる。 ｖｉｉ）粘着プラスチックフィルムに貼り付いた状態の
切片を、研究目的に応じて染色する。I) The frozen sample and the water-soluble embedding agent are put into the container part 2 of the cryoembedding container shown in FIG. ii) Holding the handle 1 of the frozen embedding container, place the container portion 2 in a refrigerant (for example, hexane at −94 ° C.) and freeze it. iii) After freezing, as shown in FIG. 2, the lower lid 3 of the container part 2 of the frozen embedding container is pulled out, and the frozen embedding block 5 is pushed out from below. iv) The taken out frozen embedding block 5 is fixed to the sample stage 6 as shown in FIG. 3, and is fixed to the sample tilting device 8 shown in FIGS. 4, 8, and 9 with the sample stage fixing screw 28. v) The adhesive plastic film produced by the above procedure is attached to the thin section of the frozen embedding block 5 as shown in FIG. vi) In this state, by performing thin sectioning with the spare blade 9 fixed to the spare blade support 11, the frozen section 40 can be taken in a state of being stuck to the adhesive plastic film. vii) The section attached to the adhesive plastic film is stained according to the purpose of the study.
【００２４】次に、上記で作製した切片を保存するため
の切片保存方法について、順に説明する。 ｉ）図１６に示すように、透明なガラス板４３に水溶性
封入剤４２（例えば、純水で希釈した３０％グリセリ
ン）を塗布する。 ｉｉ）図１７に示すように、粘着プラスチックフィルム
に貼り付いた染色した切片４１を、水溶性封入剤４２を
塗布した透明なガラス板４３に向けて置く。 ｉｉｉ）余剰の水溶性封入剤４２を、紙（例えば、ろ
紙）で吸い取る。 ｉｖ）一日以上放置し、水溶性封入剤中の水分を蒸発さ
せ、粘着プラスチックフィルムを透明なガラス板４３に
粘着させる。 ｖ）粘着後、粘着プラスチックフィルムの粘着剤未塗布
域３７を切り取り、図１８に示すように、染色した切片
４１を粘着プラスチックフィルムと透明なガラス板４３
の間に保存する。Next, a section storing method for storing the section prepared above will be described in order. i) As shown in FIG. 16, a water-soluble mounting medium 42 (for example, 30% glycerin diluted with pure water) is applied to a transparent glass plate 43. ii) As shown in FIG. 17, the stained section 41 attached to the adhesive plastic film is placed on a transparent glass plate 43 coated with a water-soluble mounting medium 42. iii) Suction excess water-soluble mounting medium 42 with paper (eg, filter paper). iv) Leave for one day or more, evaporate the water in the water-soluble mounting medium, and make the adhesive plastic film adhere to the transparent glass plate 43. v) After the adhesion, the adhesive-uncoated area 37 of the adhesive plastic film is cut off, and as shown in FIG.
Save between.
【００２５】上記の手順により、凍結した試料（例え
ば、未固定非脱灰の硬組織、実験動物の全身）から、組
織構造が良好に保たれた厚さ２〜１０μｍの凍結切片を
簡単に、しかも確実に作製することができ、その切片を
長期保存することができる。According to the above procedure, a frozen section having a thickness of 2 to 10 μm and having a good tissue structure can be easily obtained from a frozen sample (eg, unfixed, undecalcified hard tissue, whole body of an experimental animal). Moreover, it can be produced reliably, and the section can be stored for a long time.
【００２６】[0026]
【発明の効果】本発明によると、化学薬品により脱灰処
理や固定処理されてない硬組織、大きな生物試料（例え
ば、薄切面が４ｃｍ×５ｃｍの大きさ）から、厚さ２〜
１０μｍの凍結切片を簡単に、しかも確実に作製するこ
とができ、その切片を長期保存することができるように
なった。この生物組織凍結薄切方法で作製した切片は、
多くの生命科学の研究（例えば、組織学的研究、組織化
学的研究、酵素組織化学的研究、免疫組織化学的研究、
遺伝子組織化学的研究、水溶性物質の体内分布研究、微
小領域の生化学的分析）に利用することができた。According to the present invention, a hard tissue or a large biological sample (for example, a thin section having a size of 4 cm × 5 cm) that has not been decalcified or fixed by a chemical agent can be used to obtain a sample having a thickness of 2 cm.
A 10 μm frozen section could be easily and reliably prepared, and the section could be stored for a long time. Sections made by this biological tissue cryosection method are:
Many life science studies (eg, histological, histochemical, enzyme histochemical, immunohistochemical,
Gene biochemical studies, biodistribution studies of water-soluble substances, and biochemical analysis of minute areas.
【図１】本発明の一実施の形態による生物試料を凍結包
埋する凍結包埋容器の斜視図である。FIG. 1 is a perspective view of a cryo-embedding container for cryo-embedding a biological sample according to an embodiment of the present invention.
【図２】本発明の一実施の形態による凍結包埋容器と、
包埋容器から取り出した凍結包埋ブロックの斜視図であ
る。FIG. 2 shows a frozen embedding container according to one embodiment of the present invention;
It is a perspective view of the frozen embedding block taken out from the embedding container.
【図３】本発明の一実施の形態による試料台と、凍結包
埋ブロックを固定した試料台の斜視図である。FIG. 3 is a perspective view of a sample stage according to an embodiment of the present invention and a sample stage to which a frozen embedding block is fixed.
【図４】本発明の一実施の形態による試料傾斜装置、試
料台、削りかす容器、タングステンカーバイド製替刃用
支持具、替刃支持具固定台を凍結薄切装置に取付けた状
態を示す斜視図である。FIG. 4 is a perspective view showing a state in which a sample tilting device, a sample stage, a shaving container, a tungsten carbide replacement blade support, and a replacement blade support fixing base according to an embodiment of the present invention are mounted on a freeze-thinning device. FIG.
【図５】本発明の一実施の形態によるタングステンカー
バイド製替刃、タングステンカーバイド製替刃用支持
具、替刃押さえ金具を分解した状態と組み立てた状態を
示す断面図である。FIG. 5 is a cross-sectional view showing a disassembled state and an assembled state of a tungsten carbide replacement blade, a tungsten carbide replacement blade support, and a replacement blade press fitting according to an embodiment of the present invention.
【図６】本発明の一実施の形態によるタングステンカー
バイド製替刃用支持具、替刃支持具固定台、替刃支持具
固定台取付け金具を示す斜視図である。FIG. 6 is a perspective view showing a tungsten carbide replacement blade support, a replacement blade support fixture, and a replacement blade support fixture mounting bracket according to an embodiment of the present invention.
【図７】本発明の一実施の形態によるタングステンカー
バイド製替刃用支持具、替刃支持具固定台、替刃支持具
固定台取付け金具を組立てた状態を示す斜視図である。FIG. 7 is a perspective view showing a state in which a tungsten carbide replacement blade support, a replacement blade support fixture, and a replacement blade support fixture mounting bracket according to one embodiment of the present invention are assembled.
【図８】本発明の一実施の形態による試料台、試料傾斜
装置、削りかす容器の分解斜視図である。FIG. 8 is an exploded perspective view of a sample stage, a sample tilting device, and a shavings container according to an embodiment of the present invention.
【図９】本発明の一実施の形態による試料台、試料傾斜
装置、削りかす容器を組み立てた状態を示す斜視図であ
る。FIG. 9 is a perspective view showing an assembled state of a sample stage, a sample tilting device, and a shavings container according to an embodiment of the present invention.
【図１０】本発明の一実施の形態による冷凍庫に引戸式
の作業口を、従来の開き戸式開閉蓋の直下に取り付けた
斜視図である。FIG. 10 is a perspective view of a freezer according to an embodiment of the present invention, in which a sliding door type working port is attached immediately below a conventional hinged door.
【図１１】図１０のＩ−Ｉ線に沿う断面図である。11 is a sectional view taken along the line II of FIG.
【図１２】本発明の一実施の形態による粘着剤を塗布す
る領域と、プラスチックフィルムの切断個所を示す面図
である。FIG. 12 is a plan view showing a region to which the pressure-sensitive adhesive is applied according to the embodiment of the present invention and a cutting portion of the plastic film.
【図１３】図１２のＩＩ−ＩＩ線に沿う断面図である。13 is a sectional view taken along the line II-II in FIG.
【図１４】本発明の一実施の形態による凍結包埋ブロッ
クの所定面に粘着プラスチックフィルムを貼り付けた状
態の断面図である。FIG. 14 is a cross-sectional view showing a state in which an adhesive plastic film is attached to a predetermined surface of a frozen embedding block according to an embodiment of the present invention.
【図１５】本発明の一実施の形態による薄切を実施中の
凍結切片と粘着プラスチックフィルムの関係を示す断面
図である。FIG. 15 is a cross-sectional view showing a relationship between a frozen section and an adhesive plastic film during slicing according to an embodiment of the present invention.
【図１６】本発明の一実施の形態による粘着プラスチッ
クフィルムに貼り付いた染色された切片と、透明なガラ
ス板に水溶性封入剤を塗布した状態を示す断面図であ
る。FIG. 16 is a cross-sectional view showing a stained section adhered to an adhesive plastic film according to an embodiment of the present invention and a state where a water-soluble mounting medium is applied to a transparent glass plate.
【図１７】本発明の一実施の形態による粘着プラスチッ
クフィルムに貼り付いた切片を、水溶性封入剤を塗布し
た透明なガラス板上に置いた状態を示す断面図である。FIG. 17 is a cross-sectional view showing a state in which a section adhered to an adhesive plastic film according to an embodiment of the present invention is placed on a transparent glass plate coated with a water-soluble mounting medium.
【図１８】本発明の一実施の形態による粘着プラスチッ
クフィルムと透明なガラス板の間に切片を封入した状態
を示す断面図である。FIG. 18 is a cross-sectional view showing a state in which a section is sealed between an adhesive plastic film and a transparent glass plate according to an embodiment of the present invention.
１…取っ手、２…容器部、３…下蓋、４…下蓋固定用Ｌ
型金具、５…凍結包埋ブロック、６…試料台、７…凍結
包埋ブロック固定用突起、８…試料傾斜装置、９…タン
グステンカーバイド製替刃、１０…替刃押さえ金具、１
１…タングステンカーバイド製替刃用支持具、１２…替
刃支持具固定台、１３…替刃支持具固定台の取付け金
具、１４…削りかす容器、１５…廃棄口、１６…替刃押
さえ金具固定ネジ、１７…替刃固定部、１８…替刃支持
具固定ネジ、１９…替刃支持具固定台固定ネジ、２０…
Ｙ方向回転軸、２１…Ｙ方向回転軸固定金具、２２…試
料台固定金具、２３…Ｘ方向回転軸、２４…Ｘ方向回転
軸固定金具、２５…大型凍結薄切装置の試料台、２６…
Ｘ方向回転軸固定金具固定ネジ、２７…試料台傾斜固定
ネジ、２８…試料台固定ネジ、２９…開き戸式開閉蓋、
３０…透明プラスチック製引戸、３１…透明プラスチッ
ク製固定板、３２…冷気循環用送風機、３３…冷気の循
環方向、３４…粘着剤、３５…プラスチックフィルム、
３６…粘着プラスチックフィルムの切断個所、３７…粘
着剤未塗布域、３８…粘着剤塗布幅、３９…アクリル
板、４０…粘着プラスチックフィルムに貼り付いた凍結
切片、４１…染色あるいは化学処理された切片、４２…
水溶性封入剤、４３…ガラス板DESCRIPTION OF SYMBOLS 1 ... Handle, 2 ... Container part, 3 ... Lower lid, 4 ... Lower lid fixing L
Mold fitting, 5: frozen embedding block, 6: sample stage, 7: projection for fixing the frozen embedding block, 8: sample tilting device, 9: spare blade made of tungsten carbide, 10: spare blade holder, 1
DESCRIPTION OF SYMBOLS 1 ... Tungsten carbide replacement blade support, 12 ... Replacement blade support fixture, 13 ... Mounting bracket for replacement blade support fixture, 14 ... Swarf container, 15 ... Waste port, 16 ... Fix replacement blade holder Screw, 17: replacement blade fixing part, 18: replacement blade support fixing screw, 19: replacement blade support fixing base fixing screw, 20 ...
Y-direction rotation shaft, 21: Y-direction rotation shaft fixing bracket, 22: sample stage fixing bracket, 23: X-direction rotation shaft, 24: X-direction rotation shaft fixing bracket, 25: sample stage of large freeze-thinning device, 26 ...
X direction rotation axis fixing bracket fixing screw, 27: sample stage inclination fixing screw, 28: sample stage fixing screw, 29: swing door type open / close lid,
30 ... Transparent plastic sliding door, 31 ... Transparent plastic fixing plate, 32 ... Blower for cool air circulation, 33 ... Circulation direction of cool air, 34 ... Adhesive, 35 ... Plastic film,
36: Cut point of the adhesive plastic film, 37: Non-adhesive coated area, 38: Adhesive coated width, 39: Acrylic plate, 40: Frozen section stuck to the adhesive plastic film, 41: Stained or chemically treated section , 42 ...
Water-soluble mounting medium, 43 ... Glass plate
Claims (6)
生物組織凍結薄切装置の試料台に固定し、凍結切片支持
材として試料の所定面に粘着剤を塗布したプラスチック
フィルムを貼り付け、プラスチックフィルムが貼り付い
た試料を所定の刃物により、プラスチックフィルムが貼
り付いた状態で、所定の厚さに薄切する生物組織凍結薄
切方法。1. A biological sample frozen and embedded with a water-soluble embedding agent is fixed to a sample stage of a biological tissue cryosectioning apparatus, and a plastic film coated with an adhesive is adhered to a predetermined surface of the sample as a frozen section support material. A biological tissue cryosection method in which a sample to which a plastic film is attached is sliced to a predetermined thickness with a predetermined blade while the plastic film is attached.
おいて、 上記凍結切片支持材の粘着剤としてゴム系粘着剤、また
はアクリル系粘着剤を使用し、上記プラスチックフィル
ムとしてポリ塩化ビニリデンフィルムを使用し、粘着剤
を塗布したプラスチックフィルムを上記試料の大きさに
切断後、上記試料の所定面に貼り付けるようにした生物
組織凍結薄切方法。2. The method according to claim 1, wherein a rubber-based adhesive or an acrylic-based adhesive is used as an adhesive for the frozen section support material, and a polyvinylidene chloride film is used as the plastic film. A method for cryosection of a biological tissue, wherein a plastic film to which an adhesive is applied is cut into a size of the sample, and is attached to a predetermined surface of the sample.
生物組織凍結薄切装置の試料台に固定し、凍結切片支持
材として試料の所定面に粘着剤を塗布したプラスチック
フィルムを貼り付け、プラスチックフィルムが貼り付い
た試料を所定の刃物により、プラスチックフィルムを貼
り付いた状態で、所定の厚さに薄切して得た切片を、透
明なガラス板とプラスチックフィルムの間に保存する切
片保存方法。3. A biological sample frozen and embedded with a water-soluble embedding agent is fixed to a sample stage of a biological tissue cryosectioning apparatus, and a plastic film coated with an adhesive is adhered to a predetermined surface of the sample as a frozen section support material. The sample to which the plastic film is attached is attached to the plastic film with a predetermined blade, and the section obtained by slicing the sample to a predetermined thickness is stored between the transparent glass plate and the plastic film. Section storage method.
剤またはアクリル系粘着剤を塗布したプラスチックフィ
ルムに貼り付いた切片をガラス板に向けて置き、粘着剤
の粘着力を利用してプラスチックフィルムをガラス板に
粘着させて、切片を保存する切片保存方法。4. The method for storing sections according to claim 3, wherein a water-soluble encapsulant is applied on a transparent glass plate, and the sections adhered to a plastic film coated with a rubber-based adhesive or an acrylic-based adhesive are glass. A section preservation method in which a section is placed on a plate, a plastic film is adhered to a glass plate using the adhesive force of the adhesive, and the section is preserved.
包埋ブロックを作製する手段と、所定の刃物を固定する
手段と、凍結生物試料の薄切に最適な温度に冷凍庫内を
維持する手段と、生物組織凍結薄切装置に固定された凍
結試料を直交する２つの軸であるｘ軸とｙ軸に独立して
所定角度に傾斜可能な試料傾斜手段と、上記試料傾斜手
段により設定された角度で上記凍結試料を所定の厚さに
薄切可能な薄切手段を備えた生物組織凍結薄切装置。5. A means for freezing a biological tissue and a water-soluble embedding medium to prepare a frozen embedding block, a means for fixing a predetermined knife, and a method for freezing a frozen biological sample in a freezer at an optimum temperature for slicing. Means for maintaining, a sample tilting means capable of tilting the frozen sample fixed to the biological tissue cryosectioning device at a predetermined angle independently of two axes x and y which are orthogonal to each other, and the sample tilting means. A biological tissue cryo-slicing apparatus comprising slicing means capable of slicing the frozen sample to a predetermined thickness at a set angle.
おいて、 上記刃物により凍結試料を薄切するとき、ゴム系粘着剤
またはアクリル系粘着剤を塗布したプラスチックフィル
ムを凍結試料の所定面に貼り付けた状態で薄切を実行す
る生物組織凍結薄切装置。6. The biological tissue freeze-slicing device according to claim 5, wherein when the frozen sample is sliced by the blade, a plastic film coated with a rubber-based adhesive or an acrylic-based adhesive is applied to a predetermined surface of the frozen sample. A biological tissue cryosectioning device that performs slicing while attached.
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| JP2000172781 | 2000-05-06 | ||
| JP2000-172781 | 2000-05-06 | ||
| JP2000393801A JP3424126B2 (en) | 2000-05-06 | 2000-11-20 | Section storage method |
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| JP3424126B2 JP3424126B2 (en) | 2003-07-07 |
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