CN110487613A - A kind of preparation method of histotomy - Google Patents
A kind of preparation method of histotomy Download PDFInfo
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- CN110487613A CN110487613A CN201910882980.5A CN201910882980A CN110487613A CN 110487613 A CN110487613 A CN 110487613A CN 201910882980 A CN201910882980 A CN 201910882980A CN 110487613 A CN110487613 A CN 110487613A
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- tissue
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- dehydrated
- histotomy
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- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 89
- 229960004756 ethanol Drugs 0.000 claims abstract description 27
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229960000935 dehydrated alcohol Drugs 0.000 claims abstract description 17
- 239000012188 paraffin wax Substances 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 10
- 239000012024 dehydrating agents Substances 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 5
- 235000019441 ethanol Nutrition 0.000 description 23
- 208000005156 Dehydration Diseases 0.000 description 11
- 230000018044 dehydration Effects 0.000 description 11
- 238000006297 dehydration reaction Methods 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 230000008602 contraction Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000004568 cement Substances 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001112 coagulating effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000001993 wax 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
-
- 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
-
- 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
- G01N1/36—Embedding or analogous mounting of samples
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Treatments For Attaching Organic Compounds To Fibrous Goods (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The present invention discloses a kind of preparation method of histotomy, comprising the following steps: (1) tissue is fixed: tissue being successively put into two cylinder fixers and is impregnated；(2) tissue dewatering: being dehydrated tissue using automatic dehydrator, is successively carried out with the ethanol solution of low concentration to high concentration gradient tissue dewatering four times, is then dehydrated three times with dehydrated alcohol again；(3) transparent: dewatered tissue is sequentially placed into three cylinder dimethylbenzene, and the time that every cylinder impregnates is successively 0.5h, 1h, 1-2h；(4) waxdip: the tissue after transparent is sequentially placed into waxdip in four cylinder paraffin, and the time of every cylinder waxdip is successively 0.5h, 0.75h, 1h, 1-2h；(5) embedded section: embedded section is carried out to the tissue after waxdip, and obtains dry histotomy after drying.
Description
Technical field
The present invention relates to biological organization sample processing technology fields, refer specifically to a kind of preparation method of histotomy.
Background technique
In the preparation process of histotomy, tissue is fixed and dehydration is committed step.
Fixation is the most important step of tissue treatment, as soon as and the step that can not most be remedied during entire film-making, it is fixed
It is after tissue is in vitro, by handling the morphosis and position that make substance in histocyte as close possible to its animation
Process, it is therefore an objective to prevent histocyte from self-dissolving and corruption occurs, be conducive to be dehydrated.If sample is without fixation in time or fixes not
When leading to tissue automatic soup-dissolving and corruption, will be unable to reverse.
Dehydration is exactly that interior moisture will be organized to cement out using dehydrating agent, if dehydration is insufficient, residual moisture in tissue is used
The dimethylbenzene for making clarifier is not soluble in water, cannot cement out the moisture in tissue when the transparent stage, after causing
Paraffin cannot be introduced into tissue when continuous waxdip, makes tissue embrittlement, if serious dehydration deficiency can make tissue feel like jelly, leads to not cut
Complete slice out.
If waxdip method is improper, it is insufficient to also result in waxdip, can not cut out complete slice.
Summary of the invention
The present invention is intended to provide a kind of preparation method of histotomy.
Technical scheme is as follows:
A kind of preparation method of histotomy, comprising the following steps:
(1) tissue is fixed: tissue being successively put into two cylinder fixers and is impregnated；
(2) tissue dewatering: being dehydrated tissue using automatic dehydrator, with the ethanol solution of low concentration to high concentration gradient according to
Then secondary progress tissue dewatering four times is dehydrated three times with dehydrated alcohol again；
(3) transparent: dewatered tissue is sequentially placed into three cylinder dimethylbenzene, displaces the dehydrating agent in tissue, every cylinder impregnate when
Between be successively 0.5h, 1h, 1-2h；
(4) waxdip: the tissue after transparent is sequentially placed into waxdip in four cylinder paraffin, the time of every cylinder waxdip be successively 0.5h,
0.75h,1h,1-2h；
(5) embedded section: embedded section is carried out to the tissue after waxdip, and obtains dry histotomy after drying.
The neutral formalin solution that fixer described in step (1) is 4%.
Step (2) low concentration to high concentration gradient ethanol solution volumetric concentration be followed successively by 75%, 85%, 95%,
95%, each volumetric concentration ethanol solution is 1.5-2h to the dewatering time of tissue, and each dehydrated alcohol is dehydrated 1h.
Four waxdip processes described in step (4) carry out in 60-68 DEG C of water bath.
Step (3) can also replace dimethylbenzene with environment friendly transparent agent in addition to making clarifier using dimethylbenzene.
The beneficial effects of the present invention are:
1. preparation method of the present invention, since the ethanol solution using low concentration to high concentration gradient carries out four dehydrations,
Tissue contracts are avoided, are conducive to be dehydrated tissue completely, it is subsequent to be dehydrated three times with dehydrated alcohol again, it is ensured that tissue dehydration completely,
And then ensure subsequent transparent and waxdip quality, avoid tissue from occurring when leading to slice since dehydration is insufficient broken and empty
The phenomenon that hole, keeps histotomy complete.
2. the present invention is fixed tissue with two cylinder fixers, it is ensured that can fully keep tissue morphology, make albumen
Matter, nucleic acid molecule are solidified in cell in-situ to be precipitated, and prevents desmoenzyme to the decomposition of protein, make it is intracellular it is various at
Divide and completely remains behind.
3. the waxdip time of the present invention is gradually incremented by, waxdip is abundant, may advantageously facilitate the integrality of slice.
Specific embodiment
The present invention is further illustrated below.
Embodiment 1
A kind of preparation method of histotomy, comprising the following steps:
(1) tissue is fixed: tissue being successively put into two cylinder fixers and is impregnated, the neutral formalin that the fixer is 4% is molten
Liquid；
(2) tissue dewatering: being dehydrated tissue using automatic dehydrator, with the ethanol solution of low concentration to high concentration gradient according to
Secondary progress tissue dewatering four times, the volumetric concentration of ethanol solution are followed successively by 75%, 85%, 95%, 95%, and each volumetric concentration ethyl alcohol is molten
Liquid is 1.5h to the dewatering time of tissue, is then dehydrated three times with dehydrated alcohol again, each dehydrated alcohol is dehydrated 1h；
(3) transparent: dewatered tissue is sequentially placed into three cylinder dimethylbenzene, displaces the dehydrating agent in tissue, every cylinder impregnate when
Between be successively 0.5h, 1h, 1h；
(4) waxdip: the tissue after transparent is sequentially placed into waxdip in four cylinder paraffin, the time of every cylinder waxdip be successively 0.5h,
0.75h, 1h, 1h, four times waxdip process carries out in 60 DEG C of water bath；
(5) embedded section: embedded section is carried out to the tissue after waxdip, and obtains dry histotomy after drying.
Embodiment 2
A kind of preparation method of histotomy, comprising the following steps:
(1) tissue is fixed: tissue being successively put into two cylinder fixers and is impregnated, the neutral formalin that the fixer is 4% is molten
Liquid；
(2) tissue dewatering: being dehydrated tissue using automatic dehydrator, with the ethanol solution of low concentration to high concentration gradient according to
Secondary progress tissue dewatering four times, the volumetric concentration of ethanol solution are followed successively by 75%, 85%, 95%, 95%, and each volumetric concentration ethyl alcohol is molten
Liquid is 1.8h to the dewatering time of tissue, is then dehydrated three times with dehydrated alcohol again, each dehydrated alcohol is dehydrated 1h；
(3) transparent: dewatered tissue is sequentially placed into three cylinder dimethylbenzene, displaces the dehydrating agent in tissue, every cylinder impregnate when
Between be successively 0.5h, 1h, 1.5h；
(4) waxdip: the tissue after transparent is sequentially placed into waxdip in four cylinder paraffin, the time of every cylinder waxdip be successively 0.5h,
0.75h, 1h, 1.5h, four times waxdip process carries out in 65 DEG C of water bath；
(5) embedded section: embedded section is carried out to the tissue after waxdip, and obtains dry histotomy after drying.
Embodiment 3
A kind of preparation method of histotomy, comprising the following steps:
(1) tissue is fixed: tissue being successively put into two cylinder fixers and is impregnated, the neutral formalin that the fixer is 4% is molten
Liquid；
(2) tissue dewatering: being dehydrated tissue using automatic dehydrator, with the ethanol solution of low concentration to high concentration gradient according to
Secondary progress tissue dewatering four times, the volumetric concentration of ethanol solution are followed successively by 75%, 85%, 95%, 95%, and each volumetric concentration ethyl alcohol is molten
Liquid is 2h to the dewatering time of tissue, is then dehydrated three times with dehydrated alcohol again, each dehydrated alcohol is dehydrated 1h；
(3) transparent: dewatered tissue is sequentially placed into three cylinder dimethylbenzene, displaces the dehydrating agent in tissue, every cylinder impregnate when
Between be successively 0.5h, 1h, 2h；
(4) waxdip: the tissue after transparent is sequentially placed into waxdip in four cylinder paraffin, the time of every cylinder waxdip be successively 0.5h,
0.75h, 1h, 2h, four times waxdip process carries out in 68 DEG C of water bath；
(5) embedded section: embedded section is carried out to the tissue after waxdip, and obtains dry histotomy after drying.
Comparative example 1
(2) tissue dewatering: being dehydrated tissue using automatic dehydrator, with the ethanol solution of high concentration to low concentration gradient according to
Secondary progress tissue dewatering four times, the volumetric concentration of ethanol solution are followed successively by 95%, 95%, 85%, 75%, and each volumetric concentration ethyl alcohol is molten
Liquid is 1.5h to the dewatering time of tissue, is then dehydrated three times with dehydrated alcohol again, each dehydrated alcohol is dehydrated 1h；
Other contents of the present embodiment are same as Example 1.
Comparative example 2
The present embodiment the difference from embodiment 1 is that:
Step (2) tissue dewatering: tissue is dehydrated using automatic dehydrator, the ethyl alcohol with low concentration to high concentration gradient is molten
Liquid successively carries out tissue dewatering four times, and the volumetric concentration of ethanol solution is followed successively by 75%, 85%, 95%, 95%, each volumetric concentration second
Alcoholic solution is 1.5h to the dewatering time of tissue；
Other contents of the present embodiment are same as Example 1.
Comparative example 3
The present embodiment the difference from embodiment 1 is that:
(4) waxdip: the tissue after transparent is sequentially placed into waxdip in four cylinder paraffin, and the time of every cylinder waxdip is 0.5h, four leachings
Wax process carries out in 60 DEG C of water bath；
Other contents of the present embodiment are same as Example 1.
Embodiment 1-3 and the preparation-obtained histotomy of comparative example 1-3 are placed on microscopically observation slice
Integrality, as a result as shown in table 1 below
Table 1
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| It cuts Piece Effect Fruit | Histotomy is complete, nothing Contraction or hardening phenomenon, Without broken or cavity | Histotomy is complete, nothing Contraction or hardening phenomenon, Without broken or cavity | Histotomy is complete, nothing Contraction or hardening phenomenon, Without broken or cavity | It is sliced imperfect, group Shrinkage phenomenon is woven with, Have broken, empty | It is sliced imperfect, group It knits ungauged regions but has Broken, cavity | It is sliced imperfect, tissue Ungauged regions, have it is broken or Cavity |
From the comparing result of table 1 it is found that comparative example 1 carries out tissue using the ethanol solution of high concentration to low concentration gradient
Dehydration, due to first of dehydration liquid excessive concentration, leads to protein coagulating, tissue contracts, dehydration is not thorough, therefore to tissue
Will lead to histocyte when being sliced has broken and cavity, is sliced imperfect.
Comparative example 2 is only dehydrated four times with the ethanol solution of low concentration to high concentration gradient, the ethanol solution of low concentration
Penetration is strong, and it is few to carry out dehydrated tissue cellular contraction using the ethanol solution of low concentration to high concentration, but due to this four times it is de-
Water is not thorough enough to tissue dewatering, and the dimethylbenzene that when clearing process uses is not soluble in water and can only replace dehydrated alcohol, but right
It is not dehydrated using dehydrated alcohol than embodiment 2, therefore the residual moisture in tissue cannot be cemented out when clearing process, it is subsequent
Paraffin can not also have broken and/or cavity completely into tissue, when causing to be sliced, be sliced imperfect.
The every cylinder waxdip time of comparative example 3 is all 0.5h, and every cylinder waxdip time is the first cylinder waxdip in embodiment 1-3
Time, waxdip time are not incremented by gradually, since waxdip is not enough, when slice the generation of incomplete waxdip it is broken and/
Or cavity, cause to be sliced imperfect.
Preparation method of the present invention takes off since the ethanol solution using low concentration to high concentration gradient carries out four times
Water avoids tissue contracts, is conducive to be dehydrated tissue completely, subsequent to be dehydrated three times with dehydrated alcohol again, it is ensured that tissue is completely de-
Water, and then ensure subsequent transparent and waxdip quality, avoid tissue from causing to be crushed when slice since dehydration is insufficient
With the phenomenon in cavity, keep histotomy complete.
The present invention is fixed tissue with two cylinder fixers, it is ensured that can fully keep tissue morphology, make protein, core
Sour macromolecular is solidified in cell in-situ to be precipitated, and prevents desmoenzyme from keeping intracellular various composition complete the decomposition of protein
It is whole to remain behind.
Claims (4)
1. a kind of preparation method of histotomy, which comprises the following steps:
(1) tissue is fixed: tissue being successively put into two cylinder fixers and is impregnated；
(2) tissue dewatering: being dehydrated tissue using automatic dehydrator, with the ethanol solution of low concentration to high concentration gradient according to
Then secondary progress tissue dewatering four times is dehydrated three times with dehydrated alcohol again；
(3) transparent: dewatered tissue is sequentially placed into three cylinder dimethylbenzene, displaces the dehydrating agent in tissue, every cylinder impregnate when
Between be successively 0.5h, 1h, 1-2h；
(4) waxdip: the tissue after transparent is sequentially placed into waxdip in four cylinder paraffin, the time of every cylinder waxdip be successively 0.5h,
0.75h,1h,1-2h；
(5) embedded section: embedded section is carried out to the tissue after waxdip, and obtains dry histotomy after drying.
2. the preparation method of histotomy as described in claim 1, which is characterized in that fixer described in step (1) is 4%
Neutral formalin solution.
3. the preparation method of histotomy as described in claim 1, which is characterized in that step (2) low concentration is to highly concentrated
The volumetric concentration for spending the ethanol solution of gradient is followed successively by 75%, 85%, 95%, 95%, and each volumetric concentration ethanol solution is to tissue
Dewatering time is 1.5-2h, and each dehydrated alcohol is dehydrated 1h.
4. the preparation method of histotomy as described in claim 1, which is characterized in that four waxdip mistakes described in step (4)
Journey carries out in 60-68 DEG C of water bath.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910882980.5A CN110487613A (en) | 2019-09-18 | 2019-09-18 | A kind of preparation method of histotomy |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910882980.5A CN110487613A (en) | 2019-09-18 | 2019-09-18 | A kind of preparation method of histotomy |
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| CN110487613A true CN110487613A (en) | 2019-11-22 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110823665A (en) * | 2019-11-27 | 2020-02-21 | 李雄 | Application of n-butanol in preparation of low-toxicity dehydrating agent for dehydrating biopsy tissue specimen and preparing transparent sheet |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441541A (en) * | 2015-12-04 | 2016-03-30 | 北京医院 | Lung cancer detection quality control product and preparation method thereof |
| CN107677531A (en) * | 2017-10-11 | 2018-02-09 | 杨莉 | The complete cell lump paraffin section preparation method that malignant cell position is presented |
| CN108168969A (en) * | 2017-12-14 | 2018-06-15 | 漳州卫生职业学院 | Agar cell block and preparation method thereof |
-
2019
- 2019-09-18 CN CN201910882980.5A patent/CN110487613A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441541A (en) * | 2015-12-04 | 2016-03-30 | 北京医院 | Lung cancer detection quality control product and preparation method thereof |
| CN107677531A (en) * | 2017-10-11 | 2018-02-09 | 杨莉 | The complete cell lump paraffin section preparation method that malignant cell position is presented |
| CN108168969A (en) * | 2017-12-14 | 2018-06-15 | 漳州卫生职业学院 | Agar cell block and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110823665A (en) * | 2019-11-27 | 2020-02-21 | 李雄 | Application of n-butanol in preparation of low-toxicity dehydrating agent for dehydrating biopsy tissue specimen and preparing transparent sheet |
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Application publication date: 20191122 |