JPS644148B2 - - Google Patents
Info
- Publication number
- JPS644148B2 JPS644148B2 JP15758682A JP15758682A JPS644148B2 JP S644148 B2 JPS644148 B2 JP S644148B2 JP 15758682 A JP15758682 A JP 15758682A JP 15758682 A JP15758682 A JP 15758682A JP S644148 B2 JPS644148 B2 JP S644148B2
- Authority
- JP
- Japan
- Prior art keywords
- sample
- paraffin
- staining
- electron microscope
- pretreatment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- -1 fatty acid ester Chemical class 0.000 claims description 10
- 239000012188 paraffin wax Substances 0.000 claims description 10
- 238000010186 staining Methods 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 6
- 229920001214 Polysorbate 60 Polymers 0.000 claims description 5
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 5
- 229930195729 fatty acid Natural products 0.000 claims description 5
- 239000000194 fatty acid Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 229920000136 polysorbate Polymers 0.000 description 4
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- 230000002411 adverse Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010894 electron beam technology Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium atom Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
Description
【発明の詳細な説明】
本発明は光学顕微鏡を組み込んだ所謂複合型電
子顕微鏡を用いて試料を観察するために試料を染
色する際の前処理方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a pretreatment method for staining a sample for observation using a so-called compound electron microscope incorporating an optical microscope.
病院等において、診断のために採取された生体
組織等の試料を観察する際に、光学顕微鏡によつ
ては倍率が不充分であるため、電子顕微鏡あるい
は走査電子顕微鏡により更に高倍での観察が望ま
れる場合がある。このような場合、大量の試料の
採取が困難であつたり、好ましくなかつたりする
ため、微量な試料しか利用できず、光学顕微鏡と
電子顕微鏡あるいは走査電子顕微鏡の為に別々に
試料を用意することが困難となる。そこで、近時
電子顕微鏡あるいは走査電子顕微鏡に光学顕微鏡
を組み込んだ複合型電子顕微鏡が開発され、使用
されるようになつた。この複合型電子顕微鏡は観
察試料の量を節約することができるばかりでな
く、光学顕微鏡によつて捜し出された試料中の関
心の有る同一部分を電子顕微鏡観察できるという
大きな長所を有する。 When observing biological tissue samples collected for diagnosis in hospitals, etc., the magnification of some optical microscopes is insufficient, so it is desirable to observe at higher magnification using an electron microscope or scanning electron microscope. There may be cases where In such cases, it is difficult or undesirable to collect a large amount of sample, so only a small amount of sample can be used, and it is not possible to prepare separate samples for light microscopy and electron microscopy or scanning electron microscopy. It becomes difficult. Therefore, in recent years, an electron microscope or a compound electron microscope in which an optical microscope is incorporated into a scanning electron microscope has been developed and has come into use. This compound electron microscope not only saves the amount of specimen to be observed, but also has the great advantage of being able to observe with an electron microscope the same portion of interest in the specimen that has been found using an optical microscope.
ところで、このような複合型電子顕微鏡におい
て試料の孔学顕微鏡及び電子顕微鏡観察を行うた
めには、所謂パラフイン切片試料（即ち試料にパ
ラフイン包埋処理を施こしたものをミクロトーム
等により薄片にスライスしたもの）を染色し、観
察室の中にセツトしなければならない。 By the way, in order to perform pore microscopy and electron microscopy observation of a sample using such a compound electron microscope, a so-called paraffin section sample (i.e., a sample that has been embedded in paraffin is sliced into thin sections using a microtome or the like). specimen) and set it in the observation room.
従来においては、この染色はパラフインを除か
ないかぎり、不可能であるという前提にたつて、
スライドガラスに切片試料を載せて乾燥させ、更
にこの切片試料をガラスに接着させた後、キシレ
ンを用いてパラフインを溶かして（脱パラフイン
処理）生体組織のみにするという前処理を行つて
いた。その為、このような前処理を行つた後で
は、試料表面の凹凸が激しくなり、試料に電子線
を照射すると、表面の凹凸に基づいて信号量も変
化するため、得られた組成像は凹凸による大きな
誤差を含んだものとなつてしまう。又、組織が無
い部分では基板であるガラスあるいはカーボン等
の表面が露出してしまい、且つこの部分からの反
射電子の信号量と染色された部分からの信号量が
似かよる場合が多いので、染色した部分の形状あ
るいは存在が判別しにくいものであつた。 Conventionally, this staining was based on the premise that it was impossible unless the paraffin was removed.
A section sample was placed on a slide glass and allowed to dry, and after the section sample was adhered to the glass, pretreatment was performed by dissolving the paraffin using xylene (deparaffinization treatment) to leave only the living tissue. Therefore, after such pretreatment, the surface of the sample becomes extremely uneven, and when the sample is irradiated with an electron beam, the signal amount changes based on the surface unevenness, so the obtained composition image becomes uneven. This results in a large error due to this. In addition, in areas where there is no tissue, the surface of the substrate such as glass or carbon is exposed, and the signal amount of reflected electrons from this area is often similar to the signal amount from the stained area. It was difficult to distinguish the shape or existence of the stained part.
本発明はこのような従来の欠点を解決すべく成
されたもので、パラフイン包埋処理を施こした試
料を分断してパラフイン切片試料とした後、該試
料を染色するに際して、該切片試料を予めポリオ
キシエチレンソルビタン脂肪酸エステル又はポリ
オキシエチレンノニルフエニルエーテルのうちの
少くとも一方とエチルアルコールを含む溶液に浸
すようにしたことを特徴としている。 The present invention has been made to solve these conventional drawbacks, and after cutting a paraffin-embedded sample into paraffin section samples, when staining the sample, the section sample is It is characterized in that it is immersed in advance in a solution containing at least one of polyoxyethylene sorbitan fatty acid ester or polyoxyethylene nonyl phenyl ether and ethyl alcohol.
以下本発明の実施例を詳述する。 Examples of the present invention will be described in detail below.
まず、パラフイン切片試料を例えばカーボンコ
ーテイングあるいはインジウムコーテイングされ
たスライドガラスに載せて乾燥し、更にこのガラ
スに張り付ける。次に、35％のエチルアルコール
と0.025％のポリオキシエチレンソルビタン脂肪
酸エステル（トウイーン40）を含む水溶液に常温
で30分浸す。 First, a paraffin section sample is placed on, for example, a carbon-coated or indium-coated slide glass, dried, and then pasted on this glass. Next, it is immersed in an aqueous solution containing 35% ethyl alcohol and 0.025% polyoxyethylene sorbitan fatty acid ester (Tween 40) for 30 minutes at room temperature.
然る後、所定の染色処理を施こせば、脱パラフ
インを行なうことなく切片試料を短時間で充分染
色することができる。 Thereafter, by performing a predetermined staining process, the section sample can be sufficiently stained in a short time without deparaffinization.
次に第２の実施例を説明する。 Next, a second embodiment will be explained.
この実施例は、スライドガラスに接着した切片
試料を第１の実施例と同様に用意した後、35％の
エチルアルコールと0.025％のポリオキシエチレ
ンノニルフエニルエーテル（トウイーン40）を含
む水溶液に30分間浸す処理を行なう。 In this example, a section sample adhered to a glass slide was prepared in the same manner as in the first example, and then immersed in an aqueous solution containing 35% ethyl alcohol and 0.025% polyoxyethylene nonyl phenyl ether (Tween 40) for 30 minutes. Perform a soaking process for a minute.
このような前処理を行なつても最初の実施例と
同様に短時間で充分染色ができる。 Even with such pretreatment, sufficient staining can be achieved in a short time as in the first embodiment.
尚、ポリオキシエチレンソルビタン脂肪酸エス
テル又はポリオキシエチレンノニルフエニルエー
テルはトウイーン40のものに限らず、これらのト
ウイーン系に属するものなら（CH2CH2O）nH
のｎが10、20、60、80等でも良い。 In addition, polyoxyethylene sorbitan fatty acid ester or polyoxyethylene nonyl phenyl ether is not limited to Tween 40, but if it belongs to these Tween series, (CH 2 CH 2 O) nH
n may be 10, 20, 60, 80, etc.
上述したように本発明においては、パラフイン
切片試料の脱パラフインを行なうことなく短時間
で染色が可能となるが、この前処理に有機溶剤と
してエチルアルコールを用いているため、キシレ
ン、アセトン、トルエン等を用いる場合のように
パラフインを溶かすことが無いので、前処理を施
こしても試料の表面は完全に平坦なままであるた
め、走査電子顕微鏡観察する場合に、表面の凹凸
による影響の無い組成像を観察することができ
る。 As mentioned above, in the present invention, paraffin section samples can be stained in a short time without deparaffinization, but since ethyl alcohol is used as an organic solvent for this pretreatment, xylene, acetone, toluene, etc. Since the paraffin is not dissolved as in the case of using a sample, the surface of the sample remains completely flat even after pretreatment, so when observing with a scanning electron microscope, the composition is not affected by surface irregularities. Images can be observed.
又、パラフインの脱落により直接スライドガラ
スの表面が露出するということも無くなるため、
試料表面からの反射電子のみに基づいて判別のし
易い像を得ることができる。 In addition, the surface of the slide glass is no longer exposed directly due to the paraffin falling off.
An easily distinguishable image can be obtained based only on the reflected electrons from the sample surface.
更に本発明においては、有機溶剤として、試料
との親和性の優れたエチルアルコールを用いてお
り、且つ界面活性剤としてポリオキシエチレンソ
ルビタン脂肪酸エステル又はポリオキシエチレン
ノニルフエニルエーテルのような生化学的影響の
少い試薬を用いているため、前処理によつて試料
が悪影響を受けたり、あるいは損傷することはな
い。 Furthermore, in the present invention, ethyl alcohol, which has excellent affinity with the sample, is used as the organic solvent, and biochemical substances such as polyoxyethylene sorbitan fatty acid ester or polyoxyethylene nonyl phenyl ether are used as the surfactant. The sample is not adversely affected or damaged by the pretreatment, as less sensitive reagents are used.
Claims (1)
てパラフイン切片試料とした後、該試料を染色す
るに際して、該切片試料を予めポリオキシエチレ
ンソルビタン脂肪酸エステル又はポリオキシエチ
レンノニルフエニルエーテルのうちの少くとも一
方とエチルアルコールを含む溶液に浸すようにし
たことを特徴とする試料の染色前処理方法。1 After cutting a paraffin-embedded sample into paraffin section samples, when staining the sample, the section samples were preliminarily treated with polyoxyethylene sorbitan fatty acid ester or polyoxyethylene nonyl phenyl ether. A method for pre-staining a sample, comprising immersing the sample in a solution containing at least one side and ethyl alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15758682A JPS5946534A (en) | 1982-09-09 | 1982-09-09 | Predyeing processing method of specimen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15758682A JPS5946534A (en) | 1982-09-09 | 1982-09-09 | Predyeing processing method of specimen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5946534A JPS5946534A (en) | 1984-03-15 |
| JPS644148B2 true JPS644148B2 (en) | 1989-01-24 |
Family
ID=15652935
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15758682A Granted JPS5946534A (en) | 1982-09-09 | 1982-09-09 | Predyeing processing method of specimen |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5946534A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0653617A3 (en) * | 1993-11-12 | 1997-10-22 | Shino Junko | Fat-soluble substituting agent for substituting dehydrating agent for producing tissue preparation. |
| JP4946047B2 (en) * | 2005-12-28 | 2012-06-06 | 東洋紡績株式会社 | Particle standard reagent for formed component classifier |
| CN105241685B (en) * | 2015-08-10 | 2018-04-13 | 河南科技大学 | The preparation method of Hynobiidae animal skin microscopic tissue sections |
-
1982
- 1982-09-09 JP JP15758682A patent/JPS5946534A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5946534A (en) | 1984-03-15 |
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