CN110361246A - A kind of Histological section's colouring method - Google Patents
A kind of Histological section's colouring method Download PDFInfo
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- CN110361246A CN110361246A CN201910746962.4A CN201910746962A CN110361246A CN 110361246 A CN110361246 A CN 110361246A CN 201910746962 A CN201910746962 A CN 201910746962A CN 110361246 A CN110361246 A CN 110361246A
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- 238000000034 method Methods 0.000 title claims abstract description 52
- 238000004040 coloring Methods 0.000 title claims abstract description 44
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 132
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 78
- 238000004043 dyeing Methods 0.000 claims abstract description 76
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims abstract description 69
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 claims abstract description 63
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- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims abstract description 33
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- SEACYXSIPDVVMV-UHFFFAOYSA-L eosin Y Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 SEACYXSIPDVVMV-UHFFFAOYSA-L 0.000 claims description 16
- 238000001914 filtration Methods 0.000 claims description 16
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- 241000246358 Thymus Species 0.000 claims description 12
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- 229940050271 potassium alum Drugs 0.000 claims description 10
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 claims description 10
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 1
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims 1
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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
-
- 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
- G01N2001/302—Stain compositions
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- 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)
Abstract
The present invention provides a kind of HE colouring method, including (1) by biological tissue section, drying, dewaxing, graded ethanol aquation to water；(2) it is impregnated using celestine blue solution；(3) it is dyed using haematoxylin dye liquor；(4) water flow is rinsed to oil blackeite；(5) gradient alcohol dehydration；(6) it is dyed using eosin stain；(7) transparent using dimethylbenzene；(8) mounting.Acetic acid is added in haematoxylin dye liquor system of the present invention, and haematoxylin dyeing, after oil blackeite, directly appropriate acetic acid is added dropwise until carrying out eosin stains after 100% alcohol in progress dehydration in 50% alcoholic solution.It is innovated by the step, eosin stains are adjusted to after dehydration, guarantee that the graded ethanol of dehydration is pollution-free, are made graded ethanol that number be recycled and are dramatically increased, greatly reduce experimental cost.And the dyeing time of eosin stain is greatly shortened after adjusting, and shortens dyeing cycle, the quality of dyeing significantly improves.
Description
Technical field
The present invention relates to medical biotechnology Senile Mouse fields, and in particular to a kind of histological staining methods, i.e. haematoxylin
Eosin stains method, abbreviation HE dyeing.
Background technique
HE staining technique is currently the most widely used histological staining methods of field of medical biology, and H refers to hematoxylin
(hemaltoxylm), E refers to Yihong (Eosin).The intake of dyestuff is frequently due to the affinity difference between dyestuff and tissue,
And the coloring of selectivity.Affinity is affected by many factors, and such as attractive coulombic force, Van der Waals force, hydrogen bond etc., they are in time and sky
Between upper influence coloring effect.
In general, haematoxylin is by nuclei dyeing at black-and-blue, and Yihong is by cytoplasm and most of connective fibers
Dye different degrees of red.
Haematoxylin is by logwood (pith of haematoxylin tree) through hot water, and the process flows such as urea are refined and obtained, haematoxylin
It itself is not a kind of dyestuff, major oxidation product haematein is a kind of natural dye, and it is main to be that the dyestuff functions
Ingredient.
Haematein can be obtained by haematoxylin by two methods of autoxidation and chemical oxidation.The bush that autoxidation obtains
Uniformly dyeing liquid colouring power can maintain the long period, but since its production cycle is long, in field of biology using less.In haematoxylin
Functional component haematein is anion, if medialess stain, to the dyeing of core will not very sufficiently, to the affinity of tissue also compared with
It is weak, therefore according to the difference of the mordant added in haematoxylin, it can be divided into: alum hematoxylin, Garapa element, tungsten haematoxylin, molybdenum
Haematoxylin, lead haematoxylin and the haematoxylin without mordant.Routine hematoxylin dye liquor have Ehrlich, Mayer, Harris, Gill,
Delafield and Cole, wherein most-often used is Mayer haematoxylin.
Yihong belongs to Xanthene class dyestuff, and visible Yihong type is more in the market, but Eosin Y is practical most extensively.Make
For endochylema dyestuff, common institutional framework is most often shown with haematoxylin combination.
The staining procedure of HE dyeing is divided into two steps, and the first step is haematoxylin dyeing, and second is eosin stains.Wherein bush
Uniformly dyeing color can be divided into progressive dyeing and degeneration dyeing according to the difference of haematoxylin type.Progressive dyeing is disposably to reach
To the coloring effect of core, degeneration dyeing, which refers to, first overstains histotomy, is then broken up with acid alcohol, finally anti-again
It is blue.Yihong is unique in that: can be dyed different degrees of deep mixed pink to red after appropriate differentiation, is convenient for differentiation
The endochylema of different types of cell and different types of connective tissue and matrix.
In medicine and biological experiment, after obtaining purpose tissue, mostly selection is solid in formalin or Bu Shi fixer
Fixed, then practical alcohol saves.For certain specific substance, the preservation degree in different fixatives is usually not
Together, for example alcohol is poor to the hold capacity of protein in tissue, and also poor in terms of saving the activity of antigen and enzyme；Perhaps
It is intact after greasiness class is fixed in osmium tetroxide or bichromate, and fixed preservation is poor in formalin, in acetone
Or absorbed in fixation procedure in alcohol, therefore after alcohol fixation, lipid can not dye.Therefore the difficulty of subsequent dyeing is increased
Degree has higher challenge to the quality for improving HE picture.
The core of HE dyeing is the selection of dye liquor, and dyeing effect depends primarily on dyeing time, is organizing no physics
Under the premise of damage, the similarities and differences between control group and processing group are observed.Although haematoxylin and Yihong Soviet Union dye liquor are many kinds of,
From their formulation procedures it is not difficult to find that the two can not only be dissolved in water, it is also soluble in alcoholic solution.Sample contacts in HE dyeing course
Main solution be water and different gradients alcoholic solution, alkalescent water has certain haematoxylin dye liquors " anti-blue effect ", such as bright
Alum haematoxylin, Carzzzi haematoxylin.And Mayer haematoxylin both can be used for progressive dyeing can also be used for degeneration dyeing, especially
It is do not need aquation when needing core to redye to protrude the cytoplasmic components after specific stain, in order to avoid destroy or take off endochylema at
The coloring divided, Carzzzi haematoxylin can also be used for progressive core and redye, but need the oil blackeite in tap water.
Although the tinctorial strength and differentiation degree of eosin stains usually will appear following situations: one because that people likes is different
It is due to mercury salt is fixed, the deeply more difficult differentiation of eosin stains is abundant；Second is that when due to inverse alcohol serial dehydration, alcoholic solution
Excessively differentiation, causes Yihong to be easily eluted, and alcohol is contaminated while dyeing quality being caused to decline, and only has in histotomy red thin
There is deviation to make to dye, influence tinctorial quality in born of the same parents and eosinophil granule coloring, also result in experiment consumptive material waste；Third is that she
It is red to be used as endochylema dye liquor, it often will appear fungi growth phenomenon；Fourth is that being that haematoxylin (karyon) colours weaker phenomenon.HE dyeing
In the process, haematoxylin dyeing is primary, but will in water oil blackeite it is primary, break up in alcoholic solution it is primary, especially in alcoholic solution
Differentiation, alcohol differentiation speed is fast, non-selectivity, is difficult to control, and so as to cause the elution of haematoxylin dye liquor, core coloring is shallower, influences
Observation.Dyeing course can be eluted in dehydration link, most of Yihong, and the alcoholic solution of each gradient will appear different journeys
The pollution of degree can cause the interference in color to the dyeing of later batch, such as the coloring of muscle fibre under normal circumstances if not abandoning
For red, red blood cell is colored as Exocarpium Citri Rubrum, and cellulose is colored as deep pink, and the alcoholic solution after pollution is easy to make different tissues
Between coloring obscure, influence to observe.If abandoning, it will cause unnecessary economic losses.Finally, processed group of Yihong
Slice is knitted, gradient alcohol dehydration is and then carried out, numerous document discoveries are consulted, according to normal dewatering time, the coloring in Yihong
It can seriously be eluted, if shortening dewatering time, will lead to dehydration deficiency, will form water mist around tissue in subsequent mounting, sternly
Ghost image rings observation.
HE is most intuitive experiment side during current most basic widest histological staining methods and Experimental Research
Method.Can the therefore dyeing of fining be to prepare the premise of HE slice, and determine look for general character in generality, in general character
Look for the basis of difference.
Summary of the invention
In order to solve the above technical problems, the present invention is by adjusting the step in HE dyeing course, thus make dyeing effect into
One-step optimization can be with it should also be mentioned that the alcoholic solution of serial dehydration will no longer be contaminated by optimization dyeing procedure
It recycles practical, greatly reduces scientific research cost.
Specific technical solution of the present invention is as follows:
The first purpose of the invention is to provide a kind of celestine blue solution for HE dyeing, including celestine blue B, sulphur
Sour iron ammonium, glycerol, distilled water；
The celestine blue B, ammonium ferric sulfate, glycerol, distilled water w/v be
W/v is 2.5g:25g:50ml:450ml.；It matches, imitates in the present invention after adjusting component ratio
Fruit significantly improves.
The configuration method of the celestine blue solution are as follows: ammonium ferric sulfate is dissolved in cold distilled water, after mixing evenly plus
Enter celestine blue B and boil dissolution, glycerol is added, with preceding filtering in cold filtration.
It is added to high price molysite in celestine blue solution of the present invention, when being applied to HE dyeing, haematoxylin and cell can be enhanced
The binding force of core keeps karyon dyeing deeper.
In some specific embodiment, the system of celestine blue solution is as shown in table 1:
Table 1
A second object of the present invention is to provide it is a kind of for HE dyeing haematoxylin dye liquor, including haematoxylin, potassium alum,
Sodium iodate, citric acid, chloraldurate AR, distilled water；
The haematoxylin, potassium alum, sodium iodate, citric acid, chloraldurate AR, distilled water mass ratio are 1:50:0.2:1:
30:1000's；
The configuration method of the haematoxylin dye liquor are as follows: by haematoxylin, potassium alum and sodium iodate in distilled water ambient temperature overnight
After dissolution, it is added chloraldurate AR and citric acid, 5 minutes after mixture boiled, cold filtration then prepares completion, with preceding filtering.
Compared with the existing technology, the present invention joined new component in haematoxylin dye liquor system: citric acid, citric acid make
The haematoxylin weakly acidic system effectively can hinder cytosolic part to colour when being applied to HE dyeing, and coloring is made to have more specific aim,
At the same time, weakly acidic condition can be such that subsequent oil blackeite process can be completed in distilled water, it is no longer necessary to which AMMONIA TREATMENT simplifies step
Suddenly, experiment reagent is saved.In addition, chloraldurate AR is added in haematoxylin dye liquor system, enhance the coloring of karyon gold ion
Effect.
In some specific embodiment, the system of haematoxylin dye liquor is as shown in table 2:
Table 2
Third object of the present invention is to provide a kind of eosin stains for HE dyeing, including 1% Eosin Y, 1% tetrabromo
Tetrachlorofluorescein sodium B, 100% alcohol, acetic acid, thyme are fragrant；
In the eosin stain, 1% Eosin Y, 1% cyanosine B, 100% alcohol, acetic acid volume ratio be
5:1:93:1:4,1% Eosin Y, 1% cyanosine B, 100% alcohol, the mixed liquor of acetic acid and thyme are fragrant
Ratio be 250ml:1g；
The configuration method of the eosin stain are as follows: be successively mixed into 1% Eosin Y and 1% cyanosine B
After 100% alcohol, acetic acid is added, is eventually adding the dissolution of thyme sweet smell low-temperature dark, prepares within 4 months before HE Coloration experiment, it is close
It seals spare.
Compared with the existing technology, the present invention uses cyanosine B and Eosin Y 1:5 proportion, by appropriate acetic acid
Eosin stain system is added, when being applied to HE dyeing, coloring can be enhanced.Eosin stain system is added in appropriate thyme sweet smell, it can
Extend the service life in Yihong.
In some specific embodiment, the system of eosin stain is as shown in table 3:
Table 3
Fourth object of the present invention is to provide celestine blue solution above-mentioned or haematoxylin dye liquor or above-mentioned above-mentioned
Application of the eosin stain in HE dyeing.
Fifth object of the present invention is to provide a kind of Histological section HE colouring methods, and the method includes following steps
It is rapid:
(1) by biological tissue section, drying, dewaxing, graded ethanol aquation to water；
(2) it is impregnated 5 minutes using celestine blue solution；
(3) haematoxylin dye liquor dyes；According to progressive or degeneration staining method, dyeing time is determined.
(4) water flow is rinsed to oil blackeite, and the washing time is no more than 5 minutes；
(5) gradient alcohol dehydration；
(6) eosin stain dyes 3~15 seconds, it is preferred that dyeing 5 seconds.
(7) transparent twice using dimethylbenzene, 3 minutes every time；
(8) mounting.
Further, step (1) biological tissue is animal tissue, it is preferred that is animal testis tissue, liver group
It knits, renal tissue, musculature or small intestine.
Further, histotomy described in step (1) is with a thickness of 3~5 μm.
Further, dewaxing described in step (1) for using xylene soak twice, 6 minutes every time, rear 50% diformazan
Benzene impregnates 3 minutes after mixing with 50% alcohol.
Preferably, above-mentioned to impregnate dimethylbenzene used twice and number recycling respectively, the backward reuse in step (7).
Specifically, the backward is reused are as follows: step (1) dewaxing uses (first part) immersion of dimethylbenzene I 6 minutes, two
(second part) of toluene II impregnate 6 minutes, 50% dimethylbenzene impregnates 3 minutes after mixing in equal volume with 50% alcohol；Step (7) is transparent
First to impregnate 3 minutes using (second part) of dimethylbenzene II of step (1) recycling, again using the dimethylbenzene I of step (1) recycling (the
Two parts) it impregnates 3 minutes.
Further, aquation described in step (1) be successively use volumetric concentration for 100%, 100%, 90%,
80%, 70%, 50% alcohol gradient handles (being herein vol.%)；
Preferably, graded ethanol used in aquation is separately recovered, weight in the aquation or step (5) of the step (1) of Yu Houxu
It is multiple to use；
It is furthermore preferred that two part of 100% alcohol is numbered and recycled respectively, backward is reused in step (5).
Specifically, the backward is reused are as follows: step (1) aquation successively use graded ethanol 100% I (first part),
After 100% II (second part), 90%, 80%, 70%, 50% (I) aquation to water, it is separately recovered, it can be in subsequent step (1) water
Reused in change, can also step (5) ladder dehydration in, successively using step (1) recycling 50% (II),
70%, 80%, 90%, 100% II (second part), 100% I (first part) are dehydrated.Need it is clear that, step (1) aquation
Do not add 6% acetic acid in 50% alcohol (I) used, it is (every to 6% acetic acid of addition in 50% alcohol (II) in step (5) dehydration
Acetic acid and 50% alcohol by volume ratio 6ml:94ml in 100ml system).
Further, celestine blue solution described in step (2) includes celestine blue B, ammonium ferric sulfate, glycerol, distilled water,
The celestine blue B, ammonium ferric sulfate, glycerol, distilled water w/v be 2.5g:25g:50ml:450ml.
The configuration method of the celestine blue solution are as follows: ammonium ferric sulfate is dissolved in cold distilled water, after mixing evenly plus
Enter celestine blue B and boil dissolution, glycerol is added, with preceding filtering in cold filtration.
Further, haematoxylin dye liquor described in step (3) includes haematoxylin, potassium alum, sodium iodate, citric acid, hydration
Chloral AR, distilled water；
The haematoxylin, potassium alum, sodium iodate, citric acid, chloraldurate AR, distilled water mass ratio are 1:50:0.2:1:
30:1000's；
The configuration method of the haematoxylin dye liquor are as follows: by haematoxylin, potassium alum and sodium iodate in distilled water ambient temperature overnight
After dissolution, it is added chloraldurate AR and citric acid, 5 minutes after mixture boiled, cold filtration then prepares completion, with preceding filtering.
Further, dehydration described in step (5) successively to use volumetric concentration for 50%, 70%, 80%, 90%,
100%, 100% alcohol gradient is handled, and the acetic acid that concentration is 6% is added in 50% alcohol (to be had in every 100ml system
6ml acetic acid 94ml50% alcohol).Further, graded ethanol used is dehydrated to be separately recovered, it can be in subsequent step (1) water
It is reused in change, it can also the subsequent reuse in step (5).Need it is clear that, step joined 6% in (5)
50% alcohol is only reused in subsequent step (5), cannot be used in step (1)；The alcohol of other gradients was both
It is recyclable to be recycled and reused for step (1) and can also be used for step (5).
Further, eosin stain described in step (6) includes 1% Eosin Y, 1% cyanosine B, 100%
Alcohol, acetic acid, thyme are fragrant；
1% Eosin Y, 1% cyanosine B, 100% alcohol, acetic acid volume ratio be 5:1:93:1:4,
1% Eosin Y, 1% cyanosine B, 100% alcohol, the mixed liquor of acetic acid and thyme sweet smell ratio be
250ml:1g；The configuration method of the eosin stain are as follows: be successively mixed into 1% Eosin Y and 1% cyanosine B
After 100% alcohol, acetic acid is added, is eventually adding the dissolution of thyme sweet smell low-temperature dark, prepares within 4 months before HE Coloration experiment, it is close
It seals spare.
The beneficial effect of technical solution of the present invention compared with the existing technology is:
1, HE colouring method of the invention directly carries out (5) after the dyeing of step (3) haematoxylin dye liquor, the anti-basket of step (4)
50% → 100% gradient alcohol dehydration step, appropriate acetic acid is added dropwise in 50% alcoholic solution, after gradient to 100% alcohol
Eosin stains are carried out, which is that HE dyes most crucial part: firstly, the alcoholic solution environment of weak acid can remove endochylema or caryoplasm
In dyestuff, and nucleic acid complexes is made to keep complete, secondly, weak acid environment can slow down subsequent atomization, makes atomization
Controllability enhancing.It is innovated by the optimization of the step, after eosin stains are adjusted to dehydration, guarantees the gradient wine of dehydration
Essence is pollution-free, makes graded ethanol that number be recycled and dramatically increases, greatly reduces experimental cost.And eosin stain after adjusting
Dyeing time being foreshortened within 5~8 minutes 3~15 seconds by conventional, shorten dyeing cycle, simplify experimental procedure, HE dyeing effect
It is obviously improved.
2, be added to high price molysite in celestine blue solution of the invention, be applied to HE dyeing when, can enhance haematoxylin with
The binding force of nucleus keeps karyon dyeing deeper.
3, in haematoxylin dye liquor system of the invention, joined new component: citric acid, citric acid make the haematoxylin in weak acid
Property system, be applied to HE dyeing when, can effectively hinder cytosolic part to colour, make coloring have more specific aim, at the same time, weak acid ring
Border can be such that subsequent oil blackeite process can be completed in distilled water, it is no longer necessary to which AMMONIA TREATMENT simplifies step, saves experiment reagent.
In addition, chloraldurate AR is added in haematoxylin dye liquor system, enhance the coloring effect of karyon gold ion.
4, in eosin stain of the invention, cyanosine B and Eosin Y 1:5 proportion is used, by appropriate acetic acid
Eosin stain system is added, when being applied to HE dyeing, coloring can be enhanced.Eosin stain system is added in appropriate thyme sweet smell, it can
The service life for extending Yihong avoids unnecessary reagent from consuming.
Detailed description of the invention
Fig. 1 is the microexamination figure of mouse testis tissue tissue HE stained slice prepared by embodiment 4.
Specific embodiment
In order to make field technical staff better grasp technical solution of the present invention, below in conjunction with specific embodiment pair
The present invention elaborates.
It is compared using traditional HE colouring method and HE colouring method of the invention, while the dye liquor that the present invention uses is this hair
Three kinds of bright improved celestine blue solution, haematoxylin dye liquor, eosin stain dye liquor systems, and we are using a variety of different
Biological tissue carries out Experimental Comparison to the two.
1 haematoxylin dye liquor of embodiment is prepared
Preparation process: it is separately sampled by formula shown in table 4, by haematoxylin, potassium alum and sodium iodate in distilled water room temperature
It dissolves overnight, then addition chloraldurate AR and citric acid, 5 minutes after mixture boiled, cold filtration then prepares completion, before
Filtering.
Table 4
2 eosin stain of embodiment is prepared
Preparation process: it is separately sampled by formula shown in table 5,1% Eosin Y and 1% cyanosine B are successively mixed
Enter 100% alcohol, acetic acid is added, be eventually adding the dissolution of thyme sweet smell low-temperature dark, prepares within 4 months in advance, seal spare.
Table 5
3 celestine blue solution of embodiment is prepared
Preparation process: it is separately sampled by formula shown in table 6, ammonium ferric sulfate is dissolved in cold distilled water, after mixing evenly
Celestine blue is added and boils dissolution, glycerol is added, with preceding filtering in cold filtration.
Table 6
Embodiment 4 uses HE colouring method processing of the present invention with undertissue:
Testis tissue, liver organization, renal tissue, musculature and the small intestine of experimental animal are won, place before carrying out
Reason processing, the fixed above tissue, embeds, slice, and pre-treatment step is as follows:
1, fixed: sample is placed in 10% neutral formalin solution in time and impregnates after materials, determines to soak according to sample size
The time is steeped, big sample dissectible is fixed, and the young animal tissue set time is 24 hours, and adults can be appropriately extended 1~2
It, during fixed, replaces fixer 2~3 times.
2, be dehydrated: 1. 75% alcohol impregnates 3h at room temperature；2. 95% alcohol is stayed overnight；3. dehydrated alcohol I impregnates 45min；④
Dehydrated alcohol II impregnates 45min；
1. 75% alcohol impregnates 30min at 45 DEG C；2. 95% alcohol 30min；3. dehydrated alcohol I impregnates 30min；4. anhydrous
Ethyl alcohol II impregnates 30min.According to tissue size, type appropriate adjustment time.
3, transparent:
1. dimethylbenzene I impregnates 30min at room temperature；2. dimethylbenzene II impregnates 30min；
1. dimethylbenzene I impregnates 15min at 45 DEG C；2. dimethylbenzene II impregnates 15min.It is suitably adjusted according to tissue size, type
The whole time.
4, waxdip: by temperature setting in slightly above solid paraffin fusing point, 1. wax I impregnates 1.5h；2. wax II impregnates 1.5h；
5, it embeds: usually downward with maximum section；It is gently flattened with tweezers.
Histological section HE dyeing is carried out after pre-treatment:
1, it is sliced: being sliced using paraffin slicing machine, thickness is between 3um~5um, according to histocyte density, cell size
Determine that thickness, the reproductive organs slice thickness such as testis ovary are 3 μm, 4 μm of small intestine sections thickness, liver, kidney, muscle section are thick
5 μm of degree, drying.
2, dewax: be respectively adopted dimethylbenzene (I) impregnate 6 minutes, dimethylbenzene (II) impregnate 6 minutes, 50% dimethylbenzene with
50% alcohol impregnates 3 minutes after mixing in equal volume, and described to impregnate dimethylbenzene used twice and number respectively be (I), (II), time
It receives, is reused to backward in step (9).
3, volumetric concentration is successively used to divide for 100% alcohol (I) 2 minute, 100% alcohol (II) 2 minute, 90% alcohol 2
Clock, 80% alcohol 2 minutes, 70% alcohol 2 minutes, 50% alcohol, 2 minutes graded ethanol aquations were to water 2 minutes.Used in aquation
Graded ethanol be separately recovered, reuse in the step (5), it is (I) that two part of 100% alcohol used is numbered respectively, (II),
Recycling, backward is reused in step (7).
4, it is impregnated 5 minutes using celestine blue solution prepared by embodiment 1.
5, dyed using haematoxylin dye liquor prepared by embodiment 2 to right times: the reproductive organs such as testis ovary are sliced: 3
Minute, small intestine sections: 4 minutes, liver, kidney, muscle section: 5 minutes.
6, water flow is rinsed to oil blackeite about 4 minutes.
7, gradient alcohol dehydration: successively use the volumetric concentration that is recycled in step 3 for 50% (II), 70%, 80%,
90%, 100% (II), 100% (I) dehydration of alcohol, wherein to be to concentration is added in 50% alcohol (I) be 50% (II) alcohol
6% acetic acid (acetic acid and 50% alcohol by volume ratio 6ml:94ml in every 100ml system).
8, eosin stain prepared with embodiment 3 is adopted to dye 5 seconds.
9, the dimethylbenzene recycled using step 2 is transparent twice: dimethylbenzene (II) 3 minute, dimethylbenzene (I) 3 minute.
10, mounting: using neutral gum as adhesive, gently withholding, uniform and bubble-free mounting.
Embodiment 5 is organized using with the processing of tradition HE colouring method is above:
1, it is sliced: needing for purpose tissue to be switched to 3~5 μm of different-thickness according to experiment and differ, wherein testis ovary etc. is raw
Grow slices of organs with a thickness of 3 μm, 4 μm of small intestine sections thickness, liver, kidney, 5 μm of muscle section thickness.
2, (dimethylbenzene (I) 15 minute, dimethylbenzene (II) 15 minute ,+50% alcohol of 50% dimethylbenzene mix in equal volume for dewaxing
Successively replacement solution dewaxing in 2 minutes is impregnated afterwards)
3, graded ethanol (is followed successively by 100% (I) 5 minute, 100% (II) 5 minute, 85% alcohol 5 minutes, 75% alcohol 5
Minute) to distilled water 5 minutes.
4, haematoxylin dyeing of the present invention 5 minutes.
5, water flow is rinsed 10 minutes.
6,1% acidic alcohol 30 seconds (hydrochloric acid and ethyl alcohol volume ratio 1:99)
7, distilled water flushing 1 minute.
8,5% eosin stains 3 minutes or so of the invention.
9, it washes 30 seconds.
10, gradient alcohol dehydration (75% alcohol 1 minute, 85% alcohol 1 minute, 95% alcohol (I) 1 minute, 95% alcohol
(II) 1 minute, 100% alcohol (II) 3 minute, 100% alcohol (I) 3 minute).
11, the dimethylbenzene recycled using step 2 is transparent twice: dimethylbenzene (II) 3 minute, dimethylbenzene (I) 3 minute.
12, coverslip is gently withheld as adhesive using neutral gum, uniform and bubble-free mounting.
The dyeing observation of embodiment 6
Carry out microexamination to the stained slice that embodiment 4 and embodiment 5 are prepared respectively: experimental result shows the invention
Colouring method dyeing effect significantly improves.Visible cell core au bleu, kernel are clear under microscope；Cytoplasm pinkiness, dye
Chromaticness particle is high-visible.
The applicant does following comparison from coloration result of the following aspects to traditional dyeing and the new colouring method of invention:
Table 7 is to compare to the coloration result of 4 new invention colouring method of 5 traditional dyeing of embodiment and embodiment.
7 traditional dyeing of table and the comparison of new invention coloration result
From upper table as it can be seen that the dyeing effect for inventing new colouring method can more obviously observe the cell membrane and nucleus of tissue
The edge of film, core are clearly more demarcated with matter.Fig. 1 is the microexamination figure of mouse testis tissue HE stained slice prepared by embodiment 4.
For increase the invention dyeing effect persuasion and reliability, we are by the light measurement in image analysis system
Parameter carries out quantitative analysis；Light measurement parameter quantitative analysis common counter is that optical density (OD) is also known as absorbance, it refers to that light is logical
Incident intensity (Ia) before crossing certain solution or substance with pass through after light intensity (Ib) ratio logarithm, it may be assumed that OD=lg (Ia/
Ib).Existing document proves A=OD (absorbance is equal to optical density).The OD value the big, and light is absorbed that degree is bigger, the face of substance
Color is deeper；Conversely, the absorbance of substance is smaller, then the absorbed degree of light is fewer, and the color of substance is more shallow, can reflect institute
The lower situation of amount containing substance.There is different absorbing wavelengths after intracellular different chemical composition dyeing, therefore i.e. by extinction
The content of mensuration detection certain substance of cytoplasm is spent, and we further calculate nucleus and cytoplasm on this basis
Color absorbance is when applied.
First by the piece of HE of the present invention prepared by 5 tradition HE of embodiment dyeing and embodiment 4 different tissues dyed
Son respectively chooses 50, and every piece shoots 5 different visuals field and uses Image- under light microscopic unified amplification factor (10X20 times)
Pro Plus image analysis software carries out comprehensive morphological measurement analysis to image, optical density is selected, to choosing positive region to carry out
Measurement obtains result and is counted, and carries out homogeneity of variance analysis using SPSS20.0 statistical software and One-way ANOVA is examined
It tests, and thinks that difference has statistical significance when P < 0.05, statistical result adds and subtracts standard error (Mean ± SEM) table with average value
Show, measurement result such as the following table 8.
8 different tissues caryoplasm absorbance of table
By the result of statistical analysis and compare, further demonstrate that the coloring effect of colouring method of the invention is preferable,
As seen from the above table, compared with common staining method, the new method of invention can effectively enhance the nucleus coloring of most of tissues,
And effectively weakens cytoplasm coloring, keep the presentations such as endochylema in cytoplasm, muscle fibre, red blood cell and cellulose different degrees of
Deep pink is presented in red, such as muscle fibre, and Chinese red etc. is presented in red blood cell, and coloring reaches really fining dyeing clearly.Ginseng
It is visible consistent with the dyeing effect of optical microphotograph microscopic observation to examine table 7, karyon is in bright-coloured blue, and nuclear membrane kernel is high-visible,
Good, endochylema pink is differentiated, karyon endochylema is with distinct contrast.
7 conventional method of embodiment and invention dyeing new method are compared in operating procedure and time:
9. conventional method of table and invention dyeing new method comparison:
Conventional H E colouring method and HE colouring method of the present invention are compared, as a result 7 table of embodiment, 9 institute as above
Show, it can be seen that the efficiency of the HE dyeing new method dyeing course of invention significantly improves, and the used time is dyed short than conventional H E.
8 conventional method of embodiment and invention dyeing new method major experimental consumptive material comparison:
10. conventional method of table and invention dyeing new method comparison:
Note: usual each graded ethanol system is 200ml system, industrial alcohol unit price: 500ml/20 member
Conventional H E colouring method and HE colouring method of the present invention are compared, as a result shown in 8 table of embodiment as above,
By different degrees of pollution, red graded ethanol of getting the bid indicates in this state, it is necessary to replace the graded ethanol of conventional H E dyeing.
And the dehydration link for the HE dyeing new method dyeing invented, graded ethanol, which is recycled for multiple times, still to be interfered without color, is greatly reduced
The consumption of alcohol in dehydration significantly reduces experimental cost, while to guarantee that graded ethanol follows in the dehydration quality present invention
Ring access times are no more than 15 times.
The above is only the preferred embodiment of the present invention, which is not construed as limitation of the present invention, guarantor of the invention
Shield range should be defined by the scope defined by the claims..For those of ordinary skill in the art, do not departing from
In the spirit and scope of the present invention, several improvements and modifications can also be made, these improvements and modifications are also protection of the invention
Range.
Claims (10)
1. it is a kind of for HE dyeing celestine blue solution, which is characterized in that the celestine blue solution include celestine blue B,
Ammonium ferric sulfate, glycerol, distilled water；
The celestine blue B, ammonium ferric sulfate, glycerol, distilled water w/v be w/v be 2.5g:25g:
50ml:450ml.
The celestine blue solution is stirring evenly and then adding into celestine blue B and boils by the way that ammonium ferric sulfate to be dissolved in cold distilled water
Boil-off solution, cold filtration are configured and are obtained after glycerol is added, with preceding filtering.
2. it is a kind of for HE dyeing haematoxylin dye liquor, which is characterized in that the haematoxylin dye liquor include haematoxylin, potassium alum,
Sodium iodate, citric acid, chloraldurate AR, distilled water；
The haematoxylin, potassium alum, sodium iodate, citric acid, chloraldurate AR, distilled water mass ratio are 1:50:0.2:1:30:
1000；
The haematoxylin dye liquor is by after ambient temperature overnight dissolution, being added water in distilled water for haematoxylin, potassium alum and sodium iodate
Close chloral AR and citric acid, 5 minutes after mixture boiled, configuration is obtained after cold filtration, with preceding filtering.
3. a kind of eosin stain for HE dyeing, which is characterized in that the eosin stain includes 1% Eosin Y, 1% tetrabromo four
Chlorine fluorescein sodium B, 100.% alcohol, acetic acid, thyme are fragrant；
In the eosin stain, 1% Eosin Y, 1% cyanosine B, 100.% alcohol, acetic acid volume ratio be 5:
1:93:1:4,1% Eosin Y, 1% cyanosine B, 100.% alcohol, the mixed liquor of acetic acid and thyme are fragrant
Ratio be 250ml:1g；
After the eosin stain is by being successively mixed into 100% alcohol for 1% Eosin Y and 1% cyanosine B, it is added
Acetic acid configure and is obtained after being eventually adding the dissolution of thyme sweet smell low-temperature dark, prepared using first 4 months, and sealing is spare.
4. celestine blue solution described in claim 1 or haematoxylin dye liquor or as claimed in claim 3 as claimed in claim 2
Application of the eosin stain in HE dyeing.
5. a kind of Histological section HE colouring method, which is characterized in that the described method comprises the following steps:
(1) by biological tissue section, drying, dewaxing, graded ethanol aquation to water；
(2) it is impregnated 5 minutes using celestine blue solution described in claim 1；
(3) it is dyed using haematoxylin dye liquor as claimed in claim 2；
(4) water flow is rinsed to oil blackeite, and the washing time is no more than 5 minutes；
(5) gradient alcohol dehydration；
(6) it is dyed 3~15 seconds using eosin stain as claimed in claim 3, it is preferred that dyeing 5 seconds；
(7) transparent twice using dimethylbenzene, 3 minutes every time；
(8) mounting.
6. Histological section HE colouring method according to claim 5, which is characterized in that step (1) described biological tissue
For animal tissue, it is preferred that be animal testis tissue, liver organization, renal tissue, musculature or small intestine, more preferably
, the histotomy is with a thickness of 3~5 μm.
7. Histological section HE colouring method according to claim 5, which is characterized in that dewaxing described in step (1) is
Twice using xylene soak, it every time 6 minutes, is impregnated 3 minutes after being mixed in equal volume with 50.% dimethylbenzene with 50% alcohol afterwards.
8. Histological section HE colouring method according to claim 7, which is characterized in that described to impregnate used two twice
Toluene numbers recycling respectively, and backward is reused in step (7).
9. Histological section HE colouring method according to claim 5, which is characterized in that aquation described in step (1)
Successively to use volumetric concentration to handle for 100%, 100%, 90%, 80%, 70%, 50% alcohol gradient；Preferably, aquation
Graded ethanol used is separately recovered, and reuses in the aquation or step (5) of the step (1) of Yu Houxu；Preferably, described two
100% alcohol of part is numbered and is recycled respectively, and backward is reused in step (5).
10. Histological section HE colouring method according to claim 5, which is characterized in that described in step (5) dehydration be
Volumetric concentration is successively used to handle for 50%, 70%, 80%, 90%, 100%, 100% alcohol gradient, 50% alcohol
The middle acetic acid that concentration is added and is 6%；Preferably, it is dehydrated graded ethanol used and reuse is separately recovered.
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| CN113607522B (en) * | 2021-08-04 | 2023-12-01 | 深圳市美雅洁技术股份有限公司 | Continuous multi-cylinder rapid pathological processing device |
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Application publication date: 20191022 |