CN107907399A - A kind of preparation method for the mountant that can mark nucleus at the same time - Google Patents
A kind of preparation method for the mountant that can mark nucleus at the same time Download PDFInfo
- Publication number
- CN107907399A CN107907399A CN201711192562.0A CN201711192562A CN107907399A CN 107907399 A CN107907399 A CN 107907399A CN 201711192562 A CN201711192562 A CN 201711192562A CN 107907399 A CN107907399 A CN 107907399A
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- China
- Prior art keywords
- mountant
- prepared
- 1mol
- taken
- same time
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Classifications
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- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
Abstract
The invention discloses a kind of preparation method for the mountant that can mark nucleus at the same time, comprise the following steps：1) 1mol/L Trizma Base are prepared；2) 1mol/L NaH are prepared2PO4·2H2O；3) Tris phosphate buffers are prepared；4) 50% glycerine is prepared；5) mountant is prepared.The mountant that can mark nucleus at the same time prepared by the present invention, the step of eliminating dyeing and wash, can effectively simplify experiment flow, save time cost, improve work efficiency.The mountant can be used for the nuclear marker and mounting of frozen section, paraffin section, cell climbing sheet and blood smear equal samples.The mountant can be not only used for the observation of fluorescence microscope, it can also be used to the observation of laser confocal microscope.
Description
Technical field
The present invention relates to biomedicine, is specially a kind of preparation method for the mountant that can mark nucleus at the same time.
Background technology
In biomedicine experiment, it is often necessary to the specific component in cell and tissue is dyed with fluorescent dye,
Then in fluorescence microscopy Microscopic observation.Also, the fluorescent marker of nearly all sample can all dye nucleus.It is general at present
It is that the specific component in cell and nucleus are marked successively all over the experimental method used, after mountant mounting, glimmering
Viewed under light microscopy.It is required for repeatedly washing after each fluorescent marker, complex steps, also expend many times.Due to glimmering
Light is easily quenched, and the fluorescent marker operating time is more short better, thus it is necessary find it is a kind of shorten fluorescent staining method.
The content of the invention
It is an object of the invention to overcome above-mentioned background technology difficult, there is provided a kind of mountant that can mark nucleus at the same time
Preparation method.
To reach above-mentioned purpose, the technical solution that uses for：
A kind of preparation method for the mountant that can mark nucleus at the same time, comprises the following steps：
1) prepares 1mol/L Trizma Base：12.11g Trizma Base are taken to be dissolved in 100ml deionized waters, it is spare；
2) prepares 1mol/L NaH2PO4·2H2O：Take 15.62g NaH2PO4·2H2O is dissolved in 100ml deionized waters, spare；
3) prepares Tris- phosphate buffers：1mol/L Trizma Base 50ml are taken, with 1mol/L NaH2PO4·2H2O
PH to 7.6 is adjusted, it is spare；
4) prepares 50% glycerine：Glycerine 100ml is taken, 100ml deionized waters is added, fully mixes, autoclaving is spare；
5) mountants are prepared：
A. above-mentioned prepared Tris- phosphate buffers 5ml and 75ml deionized water is taken to be sufficiently mixed；
B. 20g polyvinyl alcohol is added in the mixed solution prepared to step a, is thoroughly mixed, is sealed with preservative film, is placed
60 DEG C of water-baths are stayed overnight；
C. anesin 100mg is taken to be thoroughly mixed with 50% glycerine 30ml；
D. take DAPI 0.1mg to add in the solution of step c, fully mix；
E. step d solution is slowly added in step b solution, added while mixing；
F. mixed liquid is sealed with preservative film, and wraps up lucifuge with masking foil, and 4 DEG C preserve,.
Above-mentioned steps 5）In b, the alcoholysis degree of polyvinyl alcohol is 87%-90%, molecular weight 30,000-70,000.
Having the beneficial effect that using the above scheme：The mountant that can mark nucleus at the same time prepared by the present invention, eliminates
It the step of dyeing and washing, can effectively simplify experiment flow, save time cost, improve work efficiency.The mountant can be used for
Frozen section, paraffin section, the nuclear marker and mounting of cell climbing sheet and blood smear equal samples.The mountant not only can be with
Observation for fluorescence microscope, it can also be used to the observation of laser confocal microscope.
Embodiment
The present invention is further described with reference to embodiment, but the present invention is not limited only to following embodiments, it is anticipated that this
Field technology personnel are in the case where combining the prior art, and there may be many variations for performance.
A kind of preparation method for the mountant that can mark nucleus at the same time, comprises the following steps：
1) prepares 1mol/L Trizma Base：12.11g Trizma Base are taken to be dissolved in 100ml deionized waters, it is spare；
2) prepares 1mol/L NaH2PO4·2H2O：Take 15.62g NaH2PO4·2H2O is dissolved in 100ml deionized waters, spare；
3) prepares Tris- phosphate buffers：1mol/L Trizma Base 50ml are taken, with 1mol/L NaH2PO4·2H2O
PH to 7.6 is adjusted, it is spare；
4) prepares 50% glycerine：Glycerine 100ml is taken, 100ml deionized waters is added, fully mixes, autoclaving is spare；
5) mountants are prepared：
A. above-mentioned prepared Tris- phosphate buffers 5ml and 75ml deionized water is taken to be sufficiently mixed；
B. 20g polyvinyl alcohol is added in the mixed solution prepared to step a, is thoroughly mixed, is sealed with preservative film, is placed
60 DEG C of water-baths are stayed overnight；
C. anesin 100mg is taken to be thoroughly mixed with 50% glycerine 30ml；
D. take DAPI 0.1mg to add in the solution of step c, fully mix；
E. step d solution is slowly added in step b solution, added while mixing；
F. mixed liquid is sealed with preservative film, and wraps up lucifuge with masking foil, and 4 DEG C preserve,.
Above-mentioned steps 5）In b, the alcoholysis degree of polyvinyl alcohol is 87%-90%, molecular weight 30,000-70,000.
Trizma Base are a kind of biological buffers, and Tris buffer solutions are not only widely used as the molten of nucleic acid and protein
Agent, also has many important uses.Tris is used for the protein crystal growth under condition of different pH.The low ion of Tris buffer solutions
Strength characteristics can be used for nematode（C. elegans）Lamin（lamin）Median fiber formation.Tris is also albumen
One of main component of matter electrophoretic buffer.In addition, Tris still prepares surfactant, vulcanization accelerator and some medicines
Intermediate.Tris is used also as titrimetric standard thing.
NaH2PO4·2H2Bis- hypophosphite monohydrate sodium dihydrogens of O, are two water things of sodium dihydrogen phosphate, for it is colourless to white crystals or
Crystalline powder, it is soluble easily in water, it is practically insoluble in ethanol.100 DEG C lose the crystallization water after continue to heat, then generate acid pyrophosphoric acid
Sodium.Sodium dihydrogen phosphate Chinese nickname：Sodium dihydrogen phosphate (anhydrous)；Sodium dihydrogen phosphate, it is anhydrous；Monosodium phosphate；Anhydrous phosphoric acid dihydro
Sodium；Sodium dihydrogen phosphate is anhydrous；MSP.English name：Sodium dihydrogen phosphate anhydrous, English is not
Name：Sodium phosphate monobasic dihydrate, Acid sodium phosphate, Monosodium
Orthophosphate, Primary sodium phosphate, Sodium dihydrogen phosphate, Sodium
Biphosphate, Monosodium phosphate, MSP.No. CAS：7558-80-7.Chemical formula and relative molecular mass：
NaH2PO4, 119.98, colourless crystallization or white crystalline powder.Odorless, taste is salty, acid.Hot to 100 DEG C lose whole crystallizations water,
It is scorching hot to become sodium metaphosphate.It is soluble easily in water, ethanol is practically insoluble in, its aqueous solution is in acidity.0.1mol/L aqueous solutions are at 25 DEG C
When pH be 4.5.Relative density 1.915.60 DEG C of fusing point.Commodity also have a molecular crystalline water.It is usually used in buffer, it is soft
Aqua.Manufacture calgon and sodium pyrophosphate.Measure magnesium.Improvement white medium is prepared in haploid breeding.
DAPI（4'6- diamidinos -2-phenylindone）English name：DAPI；4,6-Diamidino-2-phenylindole
dihydrochloride.Molecular formula：C16H15N5, molecular weight：277.324.Reagent available for nuclear targeting.It is that one kind can
With the blue fluorescent dyes of penetration cell film.Much stronger than DAPI itself 20 times of fluorescence can be produced after being combined with double-stranded DNA.With
EB（ethidium bromide）Compare, much higher times of the dyeing sensitivity to double-stranded DNA.DAPI dyeing is usually used in cell and withers
Detection is died, fluorescence microscope or flow cytomery are used after dyeing.DAPI be also commonly used for common nuclear targeting with
And the double-stranded DNA dyeing under some particular cases.Under fluorescence microscope, DAPI stains utilize the light of ultraviolet wavelength
Excitation.When DAPI is combined with distrand DNA, maximum absorption wave a length of 358nm, maximum emission wavelength 461nm, its diverging light
Wave-length coverage is blue to dark green containing having covered.DAPI can also be combined with RNA, but the fluorescence intensity produced too late is combined with DNA
As a result, the wave-length coverage of its diverging light about in 500nm or so.
The mountant that can mark nucleus at the same time prepared by the present invention, can be effectively simple the step of eliminating dyeing and wash
Change experiment flow, save time cost, improve work efficiency.
The mountant can be used for the nuclear marker of frozen section, paraffin section, cell climbing sheet and blood smear equal samples
And mounting.
The mountant can be not only used for the observation of fluorescence microscope, it can also be used to the observation of laser confocal microscope..
Claims (2)
1. a kind of preparation method for the mountant that can mark nucleus at the same time, it is characterized in that, comprise the following steps：
1) prepares 1mol/L Trizma Base：12.11g Trizma Base are taken to be dissolved in 100ml deionized waters, it is spare；
2) prepares 1mol/L NaH2PO4·2H2O：Take 15.62g NaH2PO4·2H2O is dissolved in 100ml deionized waters, spare；
3) prepares Tris- phosphate buffers：1mol/L Trizma Base 50ml are taken, with 1mol/L NaH2PO4·2H2O
PH to 7.6 is adjusted, it is spare；
4) prepares 50% glycerine：Glycerine 100ml is taken, 100ml deionized waters is added, fully mixes, autoclaving is spare；
5) mountants are prepared：
A. above-mentioned prepared Tris- phosphate buffers 5ml and 75ml deionized water is taken to be sufficiently mixed；
B. 20g polyvinyl alcohol is added in the mixed solution prepared to step a, is thoroughly mixed, is sealed with preservative film, is placed
60 DEG C of water-baths are stayed overnight；
C. anesin 100mg is taken to be thoroughly mixed with 50% glycerine 30ml；
D. take DAPI 0.1mg to add in the solution of step c, fully mix；
E. step d solution is slowly added in step b solution, added while mixing；
F. mixed liquid is sealed with preservative film, and wraps up lucifuge with masking foil, and 4 DEG C preserve,.
2. the preparation method of the mountant according to claim 1 that can mark nucleus at the same time, it is characterized in that：The step
5）In b, the alcoholysis degree of polyvinyl alcohol is 87%-90%, molecular weight 30,000-70,000.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711192562.0A CN107907399A (en) | 2017-11-24 | 2017-11-24 | A kind of preparation method for the mountant that can mark nucleus at the same time |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711192562.0A CN107907399A (en) | 2017-11-24 | 2017-11-24 | A kind of preparation method for the mountant that can mark nucleus at the same time |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107907399A true CN107907399A (en) | 2018-04-13 |
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| CN201711192562.0A Pending CN107907399A (en) | 2017-11-24 | 2017-11-24 | A kind of preparation method for the mountant that can mark nucleus at the same time |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105467119A (en) * | 2015-11-28 | 2016-04-06 | 上海交通大学医学院附属第三人民医院 | Method for predicting sensitivity of colorectal cancer cells to p38MAPK kinase inhibitor |
| CN105784661A (en) * | 2016-04-06 | 2016-07-20 | 北京雷根生物技术有限公司 | Mounting medium capable of reducing fluorescence decay |
| CN105891165A (en) * | 2014-11-04 | 2016-08-24 | 郝淮杰 | Method and kit for separating rare cells from peripheral blood |
| CN106370503A (en) * | 2016-08-31 | 2017-02-01 | 宁波同盛生物科技有限公司 | Organic quick drying mounting medium |
| CN106771185A (en) * | 2016-12-05 | 2017-05-31 | 湖北省肿瘤医院 | A kind of nasopharyngeal carcinoma circulating tumor cell detection kit |
-
2017
- 2017-11-24 CN CN201711192562.0A patent/CN107907399A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105891165A (en) * | 2014-11-04 | 2016-08-24 | 郝淮杰 | Method and kit for separating rare cells from peripheral blood |
| CN105467119A (en) * | 2015-11-28 | 2016-04-06 | 上海交通大学医学院附属第三人民医院 | Method for predicting sensitivity of colorectal cancer cells to p38MAPK kinase inhibitor |
| CN105784661A (en) * | 2016-04-06 | 2016-07-20 | 北京雷根生物技术有限公司 | Mounting medium capable of reducing fluorescence decay |
| CN106370503A (en) * | 2016-08-31 | 2017-02-01 | 宁波同盛生物科技有限公司 | Organic quick drying mounting medium |
| CN106771185A (en) * | 2016-12-05 | 2017-05-31 | 湖北省肿瘤医院 | A kind of nasopharyngeal carcinoma circulating tumor cell detection kit |
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| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180413 |
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| RJ01 | Rejection of invention patent application after publication |