CN101560543B - Cell staining reagent and preparation method and application thereof in cell staining - Google Patents
Cell staining reagent and preparation method and application thereof in cell staining Download PDFInfo
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- CN101560543B CN101560543B CN200810070939A CN200810070939A CN101560543B CN 101560543 B CN101560543 B CN 101560543B CN 200810070939 A CN200810070939 A CN 200810070939A CN 200810070939 A CN200810070939 A CN 200810070939A CN 101560543 B CN101560543 B CN 101560543B
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Abstract
The invention provides a cell staining reagent, comprising B.S liquid, staining liquid, rinsing liquid, and EA50 staining liquid; wherein per 400ml of B.S liquid consists of 320.0ml of methanol, 60.0ml of formol and 20.0ml of acetic acid glacial; per 210ml of staining liquid comprises 100.00mg of thiohydrazine, 87.0mg of tert-butanol liquid, 5.2ml of hydrochloric acid with the concentration of 5mol/L, 1800mg of sodium bisulfite, and the balance of distilled water; per 200ml of rising liquid comprises 1.0g of sodium metabisulfite, 2.0ml of hydrochloric acid with the concentration of 5mol/L, and198.0ml of distilled water; per 600ml of EA50 staining liquid comprises 15ml of purified water, 0.3g of bright green, 2g of water soluble eosin Y, 450ml of ethanol with volume percent of 95 percent,125ml of pure methanol, 10ml of glacial acetic acid, and 1g of phosphotungstic acid. The invention further provides the preparation method and the application of the cell staining reagent in cell staining.
Description
Technical field
The present invention relates to a kind of cell staining reagent of can be simultaneously respectively nucleus and tenuigenin being caught different colours, and compound method and the application in cell dyeing.
Technical background
Since the nearly century, along with the development of medical science, the going deep into of pathological study, the qualitative and detection by quantitative of pair cell has become domestic method medically.
The morphocytology analysis is exactly the method that pair cell carries out qualitative detection, comes pair cell to carry out qualitative analysis through the morphological feature of examining under a microscope cast-off cells, and is for referencial use for whether making further pathological analysis.For pair cell is observed; Usually want earlier whole cell to be dyeed; Make cytolemma, tenuigenin and nucleus can be easy to examine under a microscope, dyeing process commonly used has: hematoxylin eosin stain method (with organizing section statining), Papanicolaou, the Albert'stain Albert method that continues, Mai Geji dyeing etc.Several kinds of descriptive cell characteristics (like serious nucleus of very big nucleus, very big caryoplasm ratio or prilling etc.) are only arranged in the morphocytology analysis; But; These observations all are subjective; Do not have good repeatability, and the cytologist must pass through good training and could correctly detect and diagnose the illness.In addition,, exist very big difference between doctor self and the doctor, cause analyzing not accurate enough easily because this kind observation procedure subjective factor is too strong.Though the accuracy rate of diagnosis of morphocytology analysis has only about 60%, since a nearly century, still in medical science, obtained using widely.
The anti-cancer findings of the survey of many national decades confirm, can find and treatment cervical cancer early as the screening method of cervical cancer with Pap smear, and the cervical cancer mortality ratio is obviously descended.Yet,, cause Pasteur's cytology method false negative rate to reach 15%～40% owing to receive method of drawing material, smear making, the skill that dyes, read factor affecting such as sheet level.Another deficiency of Pasteur's method is the same with other conventional cytopathology diagnostic method, and the developing trend of antithetical phrase precancerous lesions of uterine cervix can't be carried out forecast assessment.
Since nineteen twenty-four Feulgen people such as (Fu Ergen) had set up the DNA-Feulgen dyeing process, this so far method was still one of main dyeing process of DNA quantitatively determined.Feulgen dyeing is the classical method that shows DNA; Its concrete reaction principle is, sample is after the Hydrogen chloride hydrolysis, and the purine bases in the dna molecular are dissociated; Thereby bring out at one of ribose and to have showed aldehyde radical; Aldehyde radical has reductive action, can combine with colourless magenta to form the red-purple compound, thereby demonstrate the distribution of DNA.Over nearly 30 years,, can catch cell image now to calculator memory, and pair cell carries out point-device measurement along with the development of digit microscope.Useful especially is to utilize the DNA of the special and stoichiometric dyeing process of DNA (i.e. dyeing is directly proportional with nucleus DNA and content) pair cell nuclear to dye, and can measure nuclear characteristic.For example feulgen's stain has obtained using widely in quantitative cytology.Utilize this method just can calculate nuclear dna content, thereby calculate the cell number ratio that is in the different cell cycles, the different cells type is detected and classifies, and aneuploid cell is detected or the like.The variation of dna content can be used as the important biomolecule of direct reflection tumor proliferation ability and learns index.Nuclear dna replication dna and cell fission are depended in cell proliferation.The essence of cell carcinogenesis is rapid infinite multiplication of cell and transfer, measures nucleus DNA content, can be used as the objective indicator of malignant tumour.Yet this method is limited by the quality of DNA fixed quality, the painted depth and mensuration system (flying-spot microscope, digital camera, application software etc.); And this method is not utilized the information of morphocytology aspect, and the information of this respect has very big effect to the diagnosis and the prediction of a lot of cases.
Therefore, in general, these two kinds of analytical procedures (qualitative and quantitative) have following problem: 1, morphocytology qualitative analysis methods a morphological feature based on cell, and dependent cells scholar's experience and skill, misdiagnosis rate is very high.2, the DNA quantitative analysis method depends on nuclear characteristic based on the special dyeing process of DNA, and any information about the morphocytology aspect can not be provided.
Up to the present, also there is not a kind of method can carry out the morphological feature qualitative analysis and the DNA quantitative analysis of cell simultaneously, yet; In the numerous disease diagnostic procedure,, need carry out qualitative and quantitative two aspects simultaneously and detect in order to improve accuracy rate; Therefore need carry out twice section and dyeing respectively, not only very bother, also increase the cost burden of sufferer; And; Qualitative result with detection by quantitative can not be corresponding one by one, such as the doctor is to find that certain has specific cell in observation morphocytology characteristic, expects the DNA quantitative analytical data of this cell so that contrast when confirming; But can't realize, because can't in the section of carrying out the DNA quantitative analysis, find corresponding cell to analyze.
Therefore, in order to carry out the detection by quantitative of morphocytology analysis and DNA simultaneously, need badly and a kind ofly can be simultaneously nucleus and tenuigenin, cytolemma be caught the cell staining reagent and the dyeing process of different colours, this case is arisen spontaneously.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can simultaneously nucleus and tenuigenin, cytolemma be caught the cell staining reagent of different colours, so that carry out the detection by quantitative of morphocytology analysis and DNA simultaneously.
A purpose more of the present invention provides the compound method of above-mentioned cell staining reagent.
The present invention also aims to provide a kind of dyeing process that uses above-mentioned cell staining reagent simultaneously nucleus and tenuigenin, cytolemma to be caught different colours.
In order to reach above-mentioned purpose, solution of the present invention is:
Cell staining reagent comprises B.S liquid, staining fluid, rinsing liquid, EA50 dye liquor, wherein:
Per 400 milliliters of B.S liquid are by 320.0 milliliters of methyl alcohol, 60.0 milliliters in formaldehyde, and 20.0 milliliters of glacial acetic acids are formulated;
Per 210 milliliters of staining fluids comprise: 100.00 milligrams of Thionin (sulphur hydrazine), and 87.0 milliliters of trimethyl carbinol liquid (analytical pure), 5.2 milliliters of 5mol/L hydrochloric acid (analytical pure), 1800 milligrams of sodium sulfite anhy 96s (analytical pure), surplus is a zero(ppm) water;
Per 200 milliliters of rinsing liquids comprise: Sodium Metabisulfite (analytical pure) 1.0 grams, 2.0 milliliters of 5mol/L hydrochloric acid (analytical pure), 198.0 milliliters of zero(ppm) water;
Per 600 milliliters of EA50 dye liquors comprise: 15 milliliters of pure water, bright green 0.3 gram, water soluble eosin Y 2 grams, 450 milliliters of 95% ethanol, 125 milliliters of pure methyl alcohol, 10 milliliters in Glacial acetic acid min. 99.5, phospho-wolframic acid 1 gram.
The compound method of above-mentioned cell staining reagent is following:
The preparation of B.S liquid: 320.0 milliliters of methyl alcohol, 60.0 milliliters in formaldehyde, 20.0 milliliters of glacial acetic acids mix;
The preparation of staining fluid: accurately take by weighing 100.00 milligrams of Thionin (sulphur hydrazine); Add 8～12 minutes postcooling to 45 of 100.0 milliliters of heated and boiled of zero(ppm) water ℃～55 ℃ (45 ℃～50 ℃ of summers); Add trimethyl carbinol liquid (analytical pure) 87.0 milliliters; Add 5.2 milliliters of 5mol/L hydrochloric acid (analytical pure) behind the mixing again, add 1800 milligrams of sodium sulfite anhy 96s (analytical pure), mix; Lucifuge stirred after 60 minutes on adding distil water to the 210 milliliter rearmounted magnetic force heating stirrer, and (stirring envrionment temperature at 33 ℃～38 ℃) filtered the back and transferred PH to 1.3～1.5 (25 ℃) to get final product; The preparation of 5mol/L hydrochloric acid: get 420 milliliters of hydrochloric acid (analytical pure) stoste adding distil water to 1000.00 milliliter;
The preparation of rinsing liquid: take by weighing Sodium Metabisulfite (analytical pure) 1.0 gram, add 5mol/L hydrochloric acid (analytical pure) 2.0 milliliters, adding distil water to 200.0 milliliter mixes, and rinsing liquid should be joined existing usefulness before use at present;
The preparation of EA50 dye liquor: a. draws 10 milliliters of pure water with suction pipe and places a clean specimen tube, with marking pen mark Yihong, draws 5 milliliters of pure water and places the clean specimen tube of another, and is bright green with the marking pen mark; B. take by weighing bright green 0.3 gram and pour in the bright green specimen tube of mark, cover lid greenly dissolves up to bright with hand rolling fully; C. take by weighing water soluble eosin Y 2 grams and pour in the specimen tube in mark Yihong, cover lid dissolves up to Yihong with hand rolling fully; D. measure 450 milliliters of 95% ethanol and pour in the clean staining jar, measure pure methyl alcohol again and pour in the cylinder for 125 milliliters; E. will dissolve bright completely green liquor and pour in the cylinder, will dissolve completely Yihong liquid and pour in the cylinder, and draw Glacial acetic acid min. 99.5 and add in the cylinder for 10 milliliters, and take by weighing phospho-wolframic acid 1 gram and add in the cylinder, and stir with splash bar and can use to mixing.
The volume and the quality of each component of cell staining reagent of more than describing only provide a kind of proportionlity, do not represent that each component of this product invention can only be used above-mentioned volume and quality.
The application of above-mentioned cell staining reagent in cell dyeing method may further comprise the steps:
The 1st step is with the sample sheet seasoning that makes.
The 2nd step; Exsiccant sample sheet was inserted the anhydrous alcohol solution internal fixing after 30 minutes, directly put into B.S liquid fixing 60 minutes, take out the back and use the distilled water wash secondary; Put into the hydrochloric acid acidifying 60 minutes (souring temperature is at 22 ℃～25 ℃) of 5mol/L again, take out the back with distilled water wash 2 times.
The 3rd step, will put into staining jar through the sample sheet of above-mentioned processing, fill it up with 67 minutes (dyeing temperature is at 22 ℃～25 ℃) of staining fluid dyeing, inclining dye liquor, with distilled water flushing 5～6 times.
In the 4th step,, use water washing again 2 to 3 times with rinsing liquid washing three times (1～2 minute for the first time, 1～2 minute second time, 1～2 minute for the third time).
In the 5th step, entering is redyed, and puts into 95% ethanol liquid and soaks 2～3 minutes.
In the 6th step, in pap staining liquid III (EA50 dye liquor), dyed endochylema 5～8 minutes.
In the 7th step, put into 95% ethanol and soaked 2～3 minutes.
The 8th step, in absolute ethyl alcohol, soak twice each 2～3 minutes.
In the 9th step, the exsiccant sheet that dewaters was put into the former fluid cylinder of YLENE transparent 30～60 seconds.
In the 10th step, the sample sheet of transparent mistake is added the deckglass sealing with neutral gum.
After adopting such scheme; Cell staining reagent provided by the invention; Can make color darker on the nuclei dyeing, and tenuigenin and cytolemma are caught more shallow color, make that cell is colored after; In the image that microscope captures, dark nucleus can both appear with the tenuigenin and the cytolemma of light color clearly.Like this, through microscopic examination, the morphological feature that in the quantitatively determined nucleus DNA, can also detect cell can improve the accuracy of cell detection and classification in order to qualitative analysis.
Description of drawings
Fig. 1 is the image that the cell after the dyed processing observes at microscopically;
Fig. 2 is that barcode is 145731 the image that sheet is examined under a microscope that gets rid of;
Fig. 3 is that barcode is 145732 the image that sheet is examined under a microscope that gets rid of;
Fig. 4 is that barcode is 145733 the image that sheet is examined under a microscope that gets rid of;
Embodiment
Preparation and the method for use of a specific embodiment to specify cell staining reagent provided by the invention below is provided, but and is not used in and limits interest field of the present invention.
The preparation of staining reagent:
The preparation of B.S liquid: 320.0 milliliters of methyl alcohol, 60.0 milliliters in formaldehyde, 20.0 milliliters of glacial acetic acids mix;
The preparation of staining fluid: accurately take by weighing 100.00 milligrams of Thionin (sulphur hydrazine); 10 minutes postcooling to 45 of 100.0 milliliters of heated and boiled of adding distil water ℃～55 ℃ (45 ℃～50 ℃ of summers); Add trimethyl carbinol liquid (analytical pure) 87.0 milliliters; Add 5.2 milliliters of 5mol/L hydrochloric acid (analytical pure) behind the mixing again, add 1800 milligrams of sodium sulfite anhy 96s (analytical pure), mix; Lucifuge stirred after 60 minutes on adding distil water to the 210 milliliter rearmounted magnetic force heating stirrer, and (stirring envrionment temperature at 33 ℃～38 ℃) filtered the back and transferred PH to 1.3～1.5 (25 ℃) to get final product; The preparation of 5mol/L hydrochloric acid: get 420 milliliters of hydrochloric acid (analytical pure) stoste adding distil water to 1000.00 milliliter;
The preparation of rinsing liquid: take by weighing Sodium Metabisulfite (analytical pure) 1.0 gram, add 5mol/L hydrochloric acid (analytical pure) 2.0 milliliters, adding distil water to 200.0 milliliter mixes, and rinsing liquid should be joined existing usefulness before use at present;
The preparation of EA50 dye liquor: (1) is drawn 10 milliliters of pure water with suction pipe and placed a clean specimen tube, with marking pen mark Yihong, draws 5 milliliters of pure water and places the clean specimen tube of another, and is bright green with the marking pen mark; (2) take by weighing bright green 0.3 gram and pour in the bright green specimen tube of mark, cover lid greenly dissolves up to bright with hand rolling fully; (3) take by weighing water soluble eosin Y 2 grams and pour in the specimen tube in mark Yihong, cover lid dissolves up to Yihong with hand rolling fully; (4) measure 450 milliliters of 95% ethanol and pour in the clean staining jar, measure pure methyl alcohol again and pour in the cylinder for 125 milliliters; (5) will dissolve bright completely green liquor and pour in the cylinder, will dissolve completely Yihong liquid and pour in the cylinder, and draw Glacial acetic acid min. 99.5 and add in the cylinder for 10 milliliters, and take by weighing phospho-wolframic acid 1 gram and add in the cylinder, and stir with splash bar and can use to mixing.
Use the cell staining reagent pair cell provided by the invention section concrete steps of handling that dye to be:
The 1st step is with the sample sheet seasoning that makes.
The 2nd step; Exsiccant sample sheet was inserted the anhydrous alcohol solution internal fixing after 30 minutes, directly put into B.S liquid fixing 60 minutes, take out the back and use the distilled water wash secondary; Put into the hydrochloric acid acidifying 60 minutes (souring temperature is at 22 ℃～25 ℃) of 5mol/L again, take out the back and use the distilled water wash secondary.
The 3rd step, will put into staining jar through the sample sheet of above-mentioned processing, fill it up with 67 minutes (dyeing temperature is at 22 ℃～25 ℃) of staining fluid dyeing, inclining dye liquor, with distilled water flushing 6 times.
In the 4th step,, use water washing again 2 to 3 times with rinsing liquid washing three times (1～2 minute for the first time, 1～2 minute second time, 1～2 minute for the third time).
In the 5th step, entering is redyed, and puts into 95% ethanol liquid and soaks 2～3 minutes.
In the 6th step, in pap staining liquid III (EA50 dye liquor), dyed endochylema 5～8 minutes.
In the 7th step, put into 95% ethanol and soaked 2～3 minutes.
The 8th step, in absolute ethyl alcohol, soak twice each 2～3 minutes.
In the 9th step, the exsiccant sheet that dewaters was put into the former fluid cylinder of YLENE transparent 30～60 seconds.
In the 10th step, the sample sheet of transparent mistake is added the deckglass sealing with neutral gum.
Dyeing process pair cell provided by the invention is redyed, so that the color that tenuigenin, cytolemma and nucleus are caught the different depths respectively is so that observation (as shown in Figure 1).
For guarantee staining reagent provided by the invention can pair cell the DNA quantitative test of nuclear impact, also carried out following two experiments:
1, choose 3 routine samples, numbering is respectively sample 1, sample 2 and sample 3; Every routine sample is done 2 and is got rid of sheet; Wherein one is used staining reagent provided by the invention to redye, and nucleus and tenuigenin are all caught color, and another is without redying; Pair cell nuclear carries out normal dyeing, draws the contrast of cell count Nomal/abnomal (normal/unusual) and integral optical density value (IOD).Referring to subordinate list 1 is through redying and the scanning result contrast that does not have through redying.
Subordinate list 1
Referring to Fig. 2 to Fig. 4, adopt dyeing process pair cell provided by the invention section to dye after, examine under a microscope, background is clear, karyon dyes mazarine, and high-visible one by one, the endochylema majority takes on a red color, and is a small amount of green.DNA detection data from subordinate list 1 through redying and not have through the detected result of redying closely, explain that the DNA detection by quantitative that this kind staining reagent can pair cell nuclear exerts an influence.
2. choose 5 routine samples, every routine sample is done 1 and is got rid of sheet, with ordinary method every routine cell section is carried out nucleus dyeing earlier, draws DI value (DNA index) through scanning; The pair cell section is moved back and is dyed then, moves back to dye and afterwards adopts dyeing process provided by the invention that every routine cell section is dyeed, and draws DI value (redying) through scanning.Subordinate list 2 is contrasts of 41 groups of nucleus DI values drawing through scanning.
Subordinate list 2
| 15 | 3.245 | 3.405 |
| 16 | 3.082 | 3.218 |
| 17 | 2.533 | 2.522 |
| 18 | 2.164 | 2.202 |
| 19 | 2.023 | 1.936 |
| 20 | 1.571 | 1.499 |
| 21 | 1.466 | 1.371 |
| 22 | 1.301 | 1.265 |
| 23 | 1.201 | 1.2 |
| 24 | 2.117 | 2.094 |
| 25 | 2.095 | 2.005 |
| 26 | 2.066 | 2 |
| 27 | 2.024 | 1.965 |
| 28 | 1.515 | 1.428 |
| 29 | 1.426 | 1.427 |
| 30 | 1.229 | 1.232 |
| 31 | 1.224 | 1.231 |
| 32 | 2.184 | 2.262 |
| 33 | 2.138 | 1.121 |
| 34 | 2.114 | 2.035 |
| 35 | 2.059 | 1.955 |
| 36 | 2.003 | 1.898 |
| 37 | 1.985 | 1.874 |
| 38 | 1.958 | 2.003 |
| 39 | 1.904 | 1.842 |
| 40 | 1.843 | 1.835 |
| 41 | 1.808 | 1.784 |
More than every group of nuclear DI value be the contrast of the nucleus DNA content (DI value) of same nucleus after conventional method dyes (dyeing) and adopts staining reagent dyeing provided by the invention (redying); Results of statistical analysis shows, no difference of science of statistics between first group and second group.
In sum; Cell staining reagent provided by the invention can make color darker on the nuclei dyeing, and tenuigenin and cytolemma are caught more shallow color, make that cell is colored after; In the image that microscope captures, dark nucleus can both appear with the tenuigenin and the cytolemma of light color clearly.And after adopting dyeing process pair cell provided by the invention to dye, examine under a microscope, nucleus presents darker color, and tenuigenin and cytolemma present more shallow color, can both clearly appear, and can pair cell the DNA quantitatively determined of nuclear impact.Like this, through microscopic examination, the morphological feature that in the quantitatively determined nucleus DNA, can also detect cell can improve the accuracy of cell detection and classification in order to qualitative analysis.
Claims (3)
1. cell staining reagent is characterized in that: comprise B.S liquid, staining fluid, rinsing liquid, EA50 dye liquor, wherein:
Per 400 milliliters of B.S liquid are by 320.0 milliliters of methyl alcohol, 60.0 milliliters in formaldehyde, and 20.0 milliliters of glacial acetic acids are formulated;
Per 210 milliliters of staining fluids comprise: 100.00 milligrams of sulphur hydrazines, and 87.0 milliliters of trimethyl carbinol liquid, 5.2 milliliters of 5mol/L hydrochloric acid, 1800 milligrams of sodium sulfite anhy 96s, surplus is a zero(ppm) water;
Per 200 milliliters of rinsing liquids comprise: Sodium Metabisulfite 1.0 grams, 2.0 milliliters of 5mol/L hydrochloric acid, 198.0 milliliters of zero(ppm) water;
Per 600 milliliters of EA50 dye liquors comprise: 15 ml distilled waters, bright green 0.3 gram, water soluble eosin Y 2 grams, 450 milliliters of 95% ethanol, 125 milliliters of pure methyl alcohol, 10 milliliters in Glacial acetic acid min. 99.5, phospho-wolframic acid 1 gram.
2. the compound method of cell staining reagent as claimed in claim 1 is characterized in that:
The preparation of B.S liquid: 320.0 milliliters of methyl alcohol, 60.0 milliliters in formaldehyde, 20.0 milliliters of glacial acetic acids mix;
The preparation of staining fluid: accurately take by weighing 100.00 milligrams of sulphur hydrazines, add 8～12 minutes postcooling to 45～55 of 100.0 milliliters of heated and boiled of zero(ppm) water ℃, add 87.0 milliliters of trimethyl carbinol liquid; Add 5.2 milliliters of 5mol/L hydrochloric acid behind the mixing again; Add 1800 milligrams of sodium sulfite anhy 96s, mix, lucifuge stirred 60～70 minutes on adding distil water to the 210 milliliter rearmounted magnetic force heating stirrer; Stir envrionment temperature at 33 ℃～38 ℃, filter the back and transfer PH to 1.3～1.5 at 25 ℃;
The preparation of rinsing liquid: take by weighing Sodium Metabisulfite 1.0 gram, add 2.0 milliliters of 5mol/L hydrochloric acid, adding distil water to 200.0 milliliter mixes;
The preparation of EA50 dye liquor: a. draws 10 ml distilled waters with suction pipe and places a clean specimen tube, with marking pen mark Yihong, draws 5 ml distilled waters and places the clean specimen tube of another, and is bright green with the marking pen mark; B. take by weighing bright green 0.3 gram and pour in the bright green specimen tube of mark, cover lid greenly dissolves up to bright with hand rolling fully; C. take by weighing water soluble eosin Y 2 grams and pour in the specimen tube in mark Yihong, cover lid dissolves up to Yihong with hand rolling fully; D. measure 450 milliliters of 95% ethanol and pour in the clean staining jar, measure pure methyl alcohol again and pour in the cylinder for 125 milliliters; E. will dissolve bright completely green liquor and pour in the cylinder, will dissolve completely Yihong liquid and pour in the cylinder, and draw Glacial acetic acid min. 99.5 and add in the cylinder for 10 milliliters, and take by weighing phospho-wolframic acid 1 gram and add in the cylinder, stir to mixing with splash bar.
3. the application of cell staining reagent as claimed in claim 1 in cell dyeing is characterized in that may further comprise the steps:
The 1st step is with the sample sheet seasoning that makes;
The 2nd step, exsiccant sample sheet was inserted the anhydrous alcohol solution internal fixing after 30 minutes, directly put into B.S liquid fixing 60 minutes; Take out the back and use the distilled water wash secondary; Put into the hydrochloric acid acidifying 60 minutes of 5mol/L again, souring temperature is at 22～25 ℃, takes out the back with distilled water wash 2 times;
The 3rd step, will put into staining jar through the sample sheet of above-mentioned processing, filled it up with staining fluid dyeing 67 minutes, dyeing temperature is at 22～25 ℃, and inclining dye liquor, with distilled water flushing 5～6 times;
In the 4th step,, 1～2 minute for the first time, 1～2 minute for the second time, 1～2 minute for the third time, use distilled water wash again 2 to 3 times with rinsing liquid washing three times;
In the 5th step, entering is redyed, and puts into 95% ethanol liquid and soaks 2～3 minutes;
In the 6th step, in the EA50 dye liquor, dyed endochylema 5～8 minutes;
In the 7th step, put into 95% ethanol and soaked 2～3 minutes;
In the 8th step, in absolute ethyl alcohol, soak twice, each 2～3 minutes;
In the 9th step, the exsiccant sheet that dewaters was put into the former fluid cylinder of YLENE transparent 30～60 seconds;
In the 10th step, the sample sheet of transparent mistake is added the deckglass sealing with neutral gum.
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| CN107603276A (en) * | 2017-09-14 | 2018-01-19 | 郑州乐业生物科技有限公司 | A kind of biological stain of stabilization and preparation method thereof |
| CN108195653A (en) * | 2017-09-16 | 2018-06-22 | 南京福怡科技发展股份有限公司 | One kind redyes staining kit and preparation and application |
| CN109187149B (en) * | 2018-09-04 | 2021-05-04 | 迈克生物股份有限公司 | Papanicolaou staining kit and staining method thereof |
| CN111337332A (en) * | 2020-03-17 | 2020-06-26 | 宁波江丰生物信息技术有限公司 | Papanicolaou staining solution EA50 and preparation method thereof |
| CN111397999B (en) * | 2020-05-09 | 2021-12-03 | 中国科学院植物研究所 | Simple method for enhancing contrast of micro-CT plant sample |
| CN113484120A (en) * | 2021-07-13 | 2021-10-08 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of Shorr staining reagent and staining method |
| CN114778256A (en) * | 2022-06-23 | 2022-07-22 | 江苏美克医学技术有限公司 | Fluorescent staining solution based on double target positions, product and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0445434B1 (en) * | 1990-03-07 | 1997-11-05 | Cytyc Corporation | Thionin stain technique |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0445434B1 (en) * | 1990-03-07 | 1997-11-05 | Cytyc Corporation | Thionin stain technique |
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