CN101241054B - Tissue block ultra-sonic dehydration method and uses thereof - Google Patents
Tissue block ultra-sonic dehydration method and uses thereof Download PDFInfo
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- CN101241054B CN101241054B CN2008100431721A CN200810043172A CN101241054B CN 101241054 B CN101241054 B CN 101241054B CN 2008100431721 A CN2008100431721 A CN 2008100431721A CN 200810043172 A CN200810043172 A CN 200810043172A CN 101241054 B CN101241054 B CN 101241054B
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Abstract
The invention relates to a preparation of tissue slice, and discloses a dehydration method of tissue block, comprising the following steps: dehydrating the fixed tissue block in gradient by ethanol aqueous solution with volume percent 70% to 100% at room temperature, using ultrasonic wave 2 to 5 times during processing of gradient dehydration by ethanol aqueous solution, 4 to 40 min each time. The invention further discloses application of the tissue block ultrasonic wave dehydration method in preparation of tissue slice. The invention reduces dehydration time of tissue block without reducing slice quality of tissue slice. The invention is especially appropriate for preparation of tissue slice with high quality.
Description
Technical field
The present invention relates to the making of histotomy, relate in particular to and utilize ultrasound wave to carry out the piece of tissue dehydration method in the histotomy manufacturing process.
Background technology
The histotomy method for making mainly contains methods such as paraffin wax flaking, quick paraffin wax flaking, freezing film-making and collodion film-making at present.But these methods all exist certain shortcoming.Time like paraffin wax flaking is long, can not satisfy the emergency treatment needs; Paraffin wax flaking is because of requiring temperature higher fast, and the slice, thin piece sharpness is relatively poor; Freezing film-making requires have liquid carbon dioxide, basic unit to be difficult to popularize; The collodion film-making is difficult to the thinness that reaches desirable, and sharpness is also poor.Therefore seeking flaking method real quickly, easy, that sharpness is good is the rapid pathological diagnosis urgent problem.And the application of ultrasound wave in film-making can effectively address the above problem.The present invention promptly is through research ultrasound wave suitable frequency, time, temperature and relevant condition in film-making dehydration, has optimized the application of ultrasound wave in paraffin wax flaking, has not only shortened histotomy Production Time, has also obtained good slice-making quality.
Summary of the invention
The purpose of this invention is to provide a kind of novel tissue block ultra-sonic dehydration method and application thereof.Tissue block ultra-sonic dehydration method of the present invention is particularly suitable for the high-quality histotomy of quick Fabrication.
The present invention has utilized hyperacoustic penetrability and cavitation effect; Passive dehydration is become dehydration initiatively, make the piece of tissue dehydration reach effect fast, but because hyperacoustic thermal effect can cause certain damage to piece of tissue; Thereby influence chipping qualities; Therefore need the relation between the conditions such as control frequency of ultrasonic, time and temperature, thereby promptly shortened dewatering time, do not reduce the slice-making quality of histotomy again.
The present invention has adopted following technical proposals:
A kind of tissue block ultra-sonic dehydration method comprises the following steps:
Under the room temperature, adopt the ethanol water gradient of concentration 70%-100% to dewater the piece of tissue after fixing, in ethanol gradient dehydration, use ultrasound wave more than 2 times, preferable use 2-5 time, each 4-40 minute.
Above-mentioned room temperature is 20 ± 5 ℃.
Above-mentioned frequency of ultrasonic scope is at 20-30KHZ, preferred 24-30KHZ.
Above-mentioned ethanol gradient dehydration is meant, adopts 70%, 80%, 95% and 100% ethanol dehydration successively.
Above-mentioned piece of tissue is fixing can to adopt conventional fixing means, as under 20 ℃, and the formaldehyde fixed with 10% 24 hours.
Preferable, above-mentioned steps 2 does, at room temperature, piece of tissue adopts 70% the of short duration well-established law dehydration of ethanol earlier, supersonic dewatering in 80%, 95% and 100% ethanol respectively then, and each supersonic dewatering time is 20-40 minute.Preferable each supersonic dewatering time is 20-30 minute.
The of short duration well-established law dewatering time of above-mentioned 70% ethanol is more than 30 minutes, during supersonic dewatering, in 80% ethanol supersonic dewatering once, twice of each supersonic dewatering in 95% and 100% the ethanol.
Better, above-mentioned steps 2 does, at room temperature; Piece of tissue adopts 70% the of short duration well-established law dehydration of ethanol earlier; With the dehydration of 80% ethanol well-established law, 95% the of short duration dehydration of ethanol well-established law is again with twice of the direct absolute ethyl alcohol dehydration of ultrasound wave again; Each supersonic dewatering time is 4-40 minute, preferred 20-30 minute.
The of short duration well-established law dewatering time of above-mentioned 70% ethanol at least 30 minutes, 80% ethanol well-established law dewatering time is 2-4 hour, 95% the of short duration dewatering time of ethanol well-established law is at least 30 minutes at every turn, dewaters twice.
The present invention also further discloses the application of above-mentioned piece of tissue supersonic dewatering method on the preparation histotomy.
The invention discloses a kind of histotomy preparation method, comprise fixing, dehydration, transparent, waxdip, section and dyeing, wherein, dehydration promptly adopts aforesaid piece of tissue supersonic dewatering method to carry out.
Preferable, above-mentioned waxdip operation is carried out under 60 ℃.
The percent concentration of above-mentioned ethanol is concentration of volume percent.
The invention has the advantages that, used room temperature to carry out piece of tissue dehydration, handled easily; Control through correlated conditions such as frequency, time and temperature that ultrasound wave is used; Make that the cripetura of total dewatering time of piece of tissue is 2 hours, compare with the conventional method dewatering time and can shorten 8-18 hour, and the section that the chipping qualities that is obtained and routine paraffin wax flaking are made is basic identical; It is more better that some places also seem; Optimized slice-making quality, shortened the time of piece of tissue in dehydration, transparent and waxdip, reduced the damage of reagent, temperature piece of tissue like method of the present invention; Dewatering and defatting is more thorough, and waxdip can reach the bath degree, and effect is fine; Section statining is more clear, the tissue and cellular contraction still less, blood vessel and compact tissue structure are more clear etc.Quick Fabrication dicing method of the present invention can also bring other benefit, as reducing reagent consumption, reduction of expenditure; Reduce and pollute, the protection staff is healthy; Also can accelerate medical services.
Description of drawings
The pig nephridial tissue slice map HE X 100 of Fig. 1 well-established law dehydration
The pig nephridial tissue slice map HE X 400 of Fig. 2 well-established law dehydration
Fig. 3 uses the pig nephridial tissue slice map HE X 100 of ultrasound wave from the dehydration of low concentration to high concentration ethanol
Fig. 4 uses the pig nephridial tissue slice map HE X 400 of ultrasound wave from the dehydration of low concentration to high concentration ethanol
Fig. 5 only uses hyperacoustic pig nephridial tissue slice map HE X 100 in the absolute ethyl alcohol dehydration
Fig. 6 only uses hyperacoustic pig nephridial tissue slice map HE X 400 in the absolute ethyl alcohol dehydration
Embodiment
Below enumerate instantiation and further specify the present invention, should be understood that instance is not to be used to limit scope of the present invention.
Embodiment 1 piece of tissue supersonic dewatering experimental study
1 materials and methods
Sample is the pig nephridial tissue.Reagent is 10% formaldehyde (V%), different concentration ethanol.CSF-1B type ultrasonic cleaner (Shanghai Ultrasonic Instrument Factory), frequency of operation are 27 ± 3KHZ, and output DC power is not more than 250W.
Cut pig kidney tissue block, every block size is 1.5cm * 1.5cm * 0.2cm, all fixes (under 20 ℃, with 10% formaldehyde fixed 24 hours) with conventional method.Divide three experimental group:
1 group, be the well-established law control group.The 5 fixing block organizations of learning from else's experience, under 20 ℃ successively once with 70% ethanol and each dehydration of 80% ethanol, each 3 hours.Use 95% ethanol dehydration secondary again, at last with the absolute ethyl alcohol secondary that dewaters, each dewatering time is 3 hours.After above-mentioned dehydration, piece of tissue inserted transparent secondary (each 1 hour) in 20 ℃ the xylene.Put into 60 ℃ paraffin waxdip secondary (each 2.5 hours).Process paraffin embedded tissues section then, HE dye (Fig. 1, Fig. 2)
2 groups, for using ultrasound wave from low concentration to high concentration ethanol dehydration group.100 pig nephridial tissues of learning from else's experience fixing are divided 20 experiment groups (2 minutes groups, 4 minutes groups, 6 minutes groups ..., to 40 minutes group totally 20 groups, every experiment group increased progressively the time of 2 minutes each supersonic dewaterings).Every experiment group is 5 pig nephridial tissues; Under 20 ℃, through the of short duration well-established law of 70% ethanol dehydration (at least 30 minutes), then supersonic dewatering is once in 80% ethanol successively; Supersonic dewatering secondary in 95% ethanol, supersonic dewatering secondary in absolute ethyl alcohol.After the dehydration, histotomy (Fig. 3, Fig. 4) is processed in, waxdip transparent with 1 group of well-established law, dyeing.
3 groups, for only in the absolute ethyl alcohol dehydration, using the ultrasound wave group.100 pig nephridial tissues of learning from else's experience fixing are divided 20 experiment groups.Every experiment group is 5 pig nephridial tissues, all under 20 ℃, and through the of short duration dehydration of 70% ethanol well-established law (at least 30 minutes), 80% ethanol well-established law dehydration (3 hours), the of short duration dehydration secondary of 95% ethanol well-established law (at least 30 minutes at every turn).Re-use the direct absolute ethyl alcohol dehydration of ultrasound wave secondary (each time is identical); Every experiment group increases progressively 2 minutes each supersonic dewatering time (from 2-40 minute; Be divided into 20 groups), after the dehydration, histotomy (Fig. 5, Fig. 6) is processed in, waxdip transparent with 1 group of well-established law, dyeing.
2 results and discussion
2.1 the result shows that 2 groups piece of tissue degree of dehydration is along with using the ultrasound wave time to be on the increase and to improve constantly.Since 20 minutes experiment groups, all piece of tissue was dewatered (comprising complete degreasing) fully.The total dewatering time of piece of tissue is the shortest to be about 2 hours.Compare with 1 group of well-established law dewatering time (10-20 hour), can shorten 8-18 hour.Process the quality of histotomy with the piece of tissue of 20 minutes minute experiment groups of experiment group to 40; 8 aspects and 1 group of comparison such as red blood cell integrated degree in the matter blood vessel between red blood cell integrated degree, renal tubule size, renal cells integrated degree and kidney from tissue contracts degree, dyeing readability, glomerulus size, glomerulus structural integrity degree, glomerular capillary, equal no significant difference.Explain and use the feasibility of ultrasound wave,, and make tissue and cell receive remarkable destruction not because of hyperacoustic use from low concentration to high concentration ethanol gradient dewatering.
2.23 group is the result show, the piece of tissue degree of dehydration is equally along with using the ultrasound wave time to be on the increase and to improve constantly.Since 4 minutes experiment groups, all piece of tissue was dewatered (comprising complete degreasing) fully.The total dewatering time of piece of tissue is the shortest to be about 4-6 hour.Compare with 1 group of well-established law dewatering time (10-20 hour), can shorten 6-14 hour.Process the quality of histotomy with the piece of tissue of minute experiment group of 4 minutes experiment groups to 40, from detecting by above-mentioned 8 aspect standards, its result also with 1 group of no significant difference.Piece of tissue is described through of short duration well-established law dehydration in early stage, the direct absolute ethyl alcohol dehydration of use ultrasound wave is feasibility equally in the certain hour scope, not because of hyperacoustic use, and makes tissue and cell receive remarkable destruction.
Use ultrasound wave 2.3 add, from total dewatering time, (3 groups the shortest are 4-6 hour to 2 groups of times length of 3 groups of ratios; 2 groups the shortest are about 2 hours).But 3 groups of needs use hyperacoustic time to significantly reduce.This makes formality to simplifying histotomy, the minimizing ultrasound wave possibly be significant to disorganization.Therefore, think that piece of tissue is done of short duration dehydration in proper order by the well-established law dehydration after, add absolute ethyl alcohol dehydration with ultrasound wave again, be better method.Be to guarantee the quality of ultrasound wave absolute ethyl alcohol dehydration, can select secondary and use the ultrasound wave time respectively to be 20-30 minute Best Times scope.
2.4 through the instruction of this experiment, those skilled in the art can understand, in the making of other histotomy, method of the present invention also is feasible again.
2.5 the section that this experiment great majority section weight and well-established law are made is basic identical; It is more better that some places also seem, more thorough like dewatering and defatting, and section statining is more clear; Tissue and cellular contraction still less, blood vessel and compact tissue structure are more clear etc. has just explained this point.
Claims (8)
1. a tissue block ultra-sonic dehydration method comprises the following steps:
Under the room temperature, it is the ethanol water gradient dehydration of 70%-100% that the piece of tissue after fixing is adopted percent by volume, in ethanol water gradient dehydration; Use ultrasound wave 2-5 time; Each 4-40 minute, said room temperature was 20 ± 5 ℃, and the frequency of ultrasonic scope is at 20-30KHZ; Said ethanol water gradient dehydration is meant, adopts the ethanol water dehydration of percent by volume 70%, 80%, 95% and 100% successively.
2. tissue block ultra-sonic dehydration method according to claim 1; It is characterized in that; The said ethanol water dehydration of adopting percent by volume 70%, 80%, 95% and 100% successively is specially: at room temperature; Piece of tissue adopts earlier 70% ethanol water dehydration, supersonic dewatering in 80%, 95% and 100% ethanol water respectively then, and each supersonic dewatering time is 20-40 minute.
3. like the said tissue block ultra-sonic dehydration method of claim 2, it is characterized in that, during said supersonic dewatering, in 80% ethanol water supersonic dewatering once, each supersonic dewatering is twice in 95% and 100% ethanol water.
4. tissue block ultra-sonic dehydration method according to claim 1; It is characterized in that; The said ethanol water dehydration of adopting percent by volume 70%, 80%, 95% and 100% successively is specially: at room temperature, piece of tissue adopts 70% ethanol water dehydration earlier, dewaters with 80% ethanol water again; 95% ethanol water dehydration, the ethanol water with ultrasound wave 100% dewaters again.
5. like the said tissue block ultra-sonic dehydration method of claim 4; It is characterized in that; Said 70% ethanol water dewatering time is more than 30 minutes, and 80% ethanol water dewatering time is 2-4 hour, and 95% ethanol water dewatering time is each more than 30 minutes; Dehydration twice, the ethanol water with ultrasound wave 100% dewaters twice again.
6. like the said tissue block ultra-sonic dehydration method of arbitrary claim among the claim 1-5, it is characterized in that said each supersonic dewatering time is 20-30 minute.
7. histotomy preparation method comprises fixing, dehydration, transparent, waxdip, section and dyeing, and wherein, dehydration adopts promptly that the said tissue block ultra-sonic dehydration method of arbitrary claim carries out among the aforementioned claim 1-6.
8. like the said histotomy preparation method of claim 7, it is characterized in that said waxdip operation is carried out under 60 ℃.
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| CN108088725A (en) * | 2017-12-28 | 2018-05-29 | 华中科技大学 | A kind of colouring method of biological tissue |
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Non-Patent Citations (3)
| Title |
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| 王诚,茹国美.超声微波快速石蜡切片技术的应用.《浙江临床医学》.2005,第7卷(第10期),第1034页. * |
| 陈谦,邹欣晔,袁忆丰,程建春.超声波声孔效应快速制取组织细胞病理切片.《中国声学学会2005年青年学术会议[CYCA 05]论文集》.2005,第500-502页. * |
| 黄永秩,卢运龙.提高超声波技术在术中病理诊断效率的方法.《左江民族医学院学报》.2007,(第5期),第809页. * |
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