CN102620966A - Biological sample preparation method applicable to ultra-thin section and fluorescence imaging - Google Patents
Biological sample preparation method applicable to ultra-thin section and fluorescence imaging Download PDFInfo
- Publication number
- CN102620966A CN102620966A CN2012100926665A CN201210092666A CN102620966A CN 102620966 A CN102620966 A CN 102620966A CN 2012100926665 A CN2012100926665 A CN 2012100926665A CN 201210092666 A CN201210092666 A CN 201210092666A CN 102620966 A CN102620966 A CN 102620966A
- Authority
- CN
- China
- Prior art keywords
- biological sample
- embedding
- gma
- ultra
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention relates to a biological sample preparation method applicable to ultra-thin section and fluorescence imaging and belongs to the technical field of biological engineering. The method comprises the following steps of: fixing biological tissue which is marked by fluorescent protein by using 4 percent paraformaldehyde; after a sample is rinsed in a phosphate buffer solution (PBS) with concentration of 0.01 mol/L, performing gradient dehydration by using an ethanol solution, wherein dehydration concentration does not exceed 95 percent; and permeating and embedding the sample by using improved glycidyl methacrylate (GMA). An embedded biological sample which is prepared by the method has hardness for continuous ultra-thin section and can keep a fluorescence signal. The method is simple, low in cost and suitable for popularization in common laboratories.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly a kind of biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging.
Background technology
Come to light and be applied to zoology as a kind of novel markings thing from the fluorescin nineties in 20th century (GFP) and derivant (XFPs) thereof, behind the fields such as botany, biology and pharmacy, life science has obtained the development of advancing by leaps and bounds.Utilize modern molecular genetic techniques, can XFPs be coupled together with any protein of interest matter,, thereby can observe the locus that is labeled albumen, move and interact then through its fluorescence of observation.Moreover, by the XFPs labelling technique, can also the various cells of specific marker, like three-dimensional spatial distribution and the annexation of neuron in brain, the 26S Proteasome Structure and Function relation of postgraduate's object is had vital role.
In the research in early days, generally adopt of the biological sample imaging of common fluorescent microscope, but because the restriction of the depth of field that formed images can only obtain the two dimensional image of slice sample to the XFPs mark.Afterwards; Copolymerization application burnt and the two-photon microscopy has significantly increased the imaging depth to fluorescent samples; But owing to receive the restriction of optical parametrics such as tissue scatter, its maximum imaging depth still can't be used to obtain the three-dimensional data of cm size mcroorganism sample still less than 1 millimeter.Over past ten years; In order to carry out the high-precision three-dimensional imaging to the large scale biological sample; A series of novel optical imaging techniques that improve sample axial resolution and imaging scope through mechanical slice have been developed both at home and abroad; Like microoptic computed tomography (SPECT) system (MOST), knife edge scanning imaging system (KESM) etc.These technology can be carried out ultra-thin section in order to guarantee biological sample, all are the embedding method that adopts Electronic Speculum basically, promptly adopt the resin embedding biological sample.
The resin that uses in the Electronic Speculum generally can be divided into two types, and one type is epikote, like Spurr and 812 etc., has hydrophobic property, needs sample to dewater fully before the use; Another kind of is acrylic resin, and like LR White, LR Gold and Lowicryl etc. have water-wet behavior, allow before the use that sample contains a certain amount of moisture.Bibliographical information is arranged, and the physico chemical factor that some are extreme is like high temperature; Strong reductant-oxidant; Strong acid and strong base reaches dehydration fully etc., all can cause the fluorescence intensity of XFPs to reduce even cancellation, so the epikote that needs sample to dewater fully is not suitable for preparing the biological sample of XFPs mark.There was the researcher to begin to attempt under condition of ultralow temperature to come the biological sample of embedding XFPs mark in recent years with hydrophilic acryl resin; Cell like GFP or RFP mark; Zebrafish embryo; Nematode or the like; The gained sample can carry out ultra-thin section and can be used to obtain fluoroscopic image; Like document " Fluorescence-integrated transmission electron microscopy images:integrating fluorescence microscopy with transmission electron microscopy. (Sims, P.A.and J.D.hardin, Methods Mol Biol 369 (2007) 291-308) ", " A Single Method for Cryofixation and Correlative Light; Electron Microscopy and Tomography of Zebrafish Embryos. (Nixon; S.J., R.I.Webb, et al.Traffic 10 (2009) 131-136) ", " Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. (Kukulski; W.; M.Schorb, et al.The Journal of Cell Biology 192 (2011) 111-119), " Protein localization in electron micrographs using fluorescence nanoscopy. (Watanabe, S.et al.Nature Methods 8 (2011) 80-84) " etc.Yet still there is following limitation in the fluorescent samples preparation method who provides in above-mentioned these documents: expensive device such as quick high-pressure frigorimeter and freezing alternative appearance have been used in (1), can't promote in common lab; (2) the quick high-pressure frigorimeter only is used to handle the tissue less than 200 micron thickness, so also can only be used to handle size less than 200 microns biological sample based on the sample preparation method of this equipment; (3) used heavy metal class immobile liquids such as acetic acid uranium or osmium tetroxide, not only had very big toxicity, but also can reduce the fluorescence of XFPs; (4) still there is a certain amount of fluorescence losses in the gained biological sample, have in addition up to more than 40%; (5) all operations all need carry out under-90 ℃ to-20 ℃ ultra-low temperature surroundings, and complex operation, has only the personnel through professional training to grasp.
In view of microoptic computed tomography (SPECT) system novel three-dimensional formation methods such as (MOST) can carry out continuous ultra-thin section and imaging to the large-sized biological sample of centimetre-sized; And be section real time imagery in slicing processes to moving; So the fluorescence signal that requires sample is than higher; And existing fluorescent samples preparation method can not satisfy preparation large scale biological sample and the requirement that keeps fluorescence fully simultaneously.Therefore, it is necessary developing a kind of technology large scale biological sample preparation method simple, that be suitable for ultra-thin section and fluorescence imaging.
Summary of the invention
The objective of the invention is provides a kind of biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging for addressing the above problem.This method can keep the fluorescence signal of biological sample, and with low cost, and operation steps is simple, is adapted at common laboratory and promotes the use of.
The technical scheme that the present invention adopted is:
A kind of biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging may further comprise the steps:
(1) biological sample being used the pre-cooled concentration of frozen water is fixing 6～24 hours of 4% paraformaldehyde immobile liquid;
(2) biological sample that step (1) is obtained cleaned 3～24 hours with the pre-cooled rinsing liquid of frozen water, changed fresh rinsing liquid 3 times therebetween;
(3) it is to carry out gradient dehydration, each 15 minutes to 2 hours in 50%, 70% and 95% ethanolic solution that the biological sample that step (2) is obtained is put into the pre-cooled concentration of frozen water successively;
(4) it is 70% that the biological sample that step (3) is obtained is put into the pre-cooled concentration of frozen water successively; Carry out gradient penetration in the water-soluble embedding liquid of 85% and 100% glycolmethacrylate (GMA); Each infiltration 1～3 hour; Put into fresh concentration subsequently once more and be 100% the water-soluble embedding liquid of GMA and permeated 6～12 hours, in the water-soluble embedding liquid of the GMA of pre-polymerization, permeated 1～3 day at last;
(5) biological sample that step (4) is obtained is put into the embedding mould oxygen barrier embedding of the water-soluble embedding liquid of the GMA that is full of pre-polymerization, inserts in 58 ℃～60 ℃ baking ovens polyase 13 then 6～60 hours.
Preferably, the immobile liquid in the said step (1) is 4% paraformaldehyde powder by concentration, and 2.5% sucrose and 0.01MOL/L PBS are formulated.
Further, said step (1) if in the biological sample tissue responsive to anoxic, can pass through the animal hearts perfusion fixation earlier, taking out required tissue again, to put into the pre-cooled concentration of frozen water be that 4% immobile liquid is fixed.
Further, in the said step (1) if the back was fixing again after if the size of biological sample, was cut into the piece of tissue of 2 centimeter square earlier excessive.
Preferably, rinsing liquid is that 0.38% glycocoll, 2.5% sucrose and 0.01MOL/L PBS are formulated by concentration in the said step (2).
Preferably, said step (4) concentration be 100% the water-soluble embedding liquid of GMA by 67 gram GMA monomers, 3 gram water, 30 gram butyl methacrylates and 0.6 gram benzoyl peroxide are formulated.
Further, be added with NaOH solution in the water-soluble embedding liquid of the GMA in said step (4) and (5), the pH value that makes the water-soluble embedding liquid of GMA is 8～9.5.
Because the water-soluble embedding liquid of GMA is low acid, can reduce the fluorescence of biological sample, in order to improve fluorescence intensity, regulate its pH value so add NaOH solution.Add NaOH amount confirm according to the corresponding pH value of different fluorescin maximum fluorescence intensities.
Preferably; The water-soluble embedding liquid making method of the GMA of the pre-polymerization in said step (4) and (5) is: gets concentration and is 100% the water-soluble embedding liquid of GMA and put into conical flask, seal the back with pan paper and bungee and insert a mercury thermometer, and a contact bottle end; Conical flask is put into heated and stirred on the heating magnetic stirring apparatus; When mercury thermometer indicated temperature to 115 ℃～120 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, reduce to the temperature of ice bath until solution temperature.
Preferably, the embedding mould is the embedding mould with oxygen barrier function in the said step (5), and said embedding mould with oxygen barrier function can be gel capsule and BEEM capsule.
Further, if there is not the embedding mould of oxygen barrier function, also can then the embedding mould be put into airtight box in said (5), put into the baking oven polymerization at last again at embedding biological sample under the oxygen free condition.
Preferably, said biological sample can be the animal tissue of any fluorescent protein labeling.
The present invention has the following advantages:
(1) biological sample of this method ability embedding cm size; (2) this method can satisfy the requirement that sample hardness is fit to continuous ultra-thin section; (3) this method can keep the fluorescence signal of sample fully; (4) method step is simple, and is with low cost, and the instrument of use, experiment conditions such as reagent and temperature all are the routine configurations of common lab, and those skilled in the art can grasp.The biological sample of gained embedding combines ultra-thin section three-dimensional imaging technology, can be used for obtaining the meticulous three-dimensional fluorescence distribution of fluorescent protein labeling tissue, carries out dependency structure and functional study then.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed explanation.
1 micron section fluoroscopic image of the full brain sample of adult Thy1-eYFP-H mouse of a kind of biological sample preparation method preparation that is applicable to ultra-thin section and fluorescence imaging that Fig. 1 provides for the embodiment of the invention 1.
0.5 micron section fluoroscopic image of 1 millimeter thickness brain of adult Thy1-eYFP-H mouse sheet of a kind of biological sample preparation method preparation that is applicable to ultra-thin section and fluorescence imaging that Fig. 2 provides for the embodiment of the invention 2.
1 micron section fluoroscopic image of the full brain sample of GFP-M transgenic mice childhood of a kind of biological sample preparation method preparation that is applicable to ultra-thin section and fluorescence imaging that Fig. 3 provides for the embodiment of the invention 3.
Embodiment
Embodiment 1
After the Thy1-eYFP-H transgenic mice of growing up is anaesthetized with 1% yellow Jackets; 0.01MOL/L PBS solution through 37 ℃ of heart left ventricle perfusions 3 minutes; After treating that blood is rinsed well, pouring into the pre-cooled concentration of frozen water immediately is 4% paraformaldehyde immobile liquid, and continues 1 hour.Then, take out the full brain of mouse, putting into the pre-cooled concentration of frozen water once more is fixing 24 hours of 4% paraformaldehyde immobile liquid back.Wherein the prescription of immobile liquid is 4 gram paraformaldehyde powder, 2.5 gram sucrose and 100 milliliters of 0.01MOL/L PBS solution.
Behind fixing the end, full brain sample is put into the pre-cooled rinsing liquid rinsing of frozen water 24 hours, more renew liquid 3 times therebetween, thoroughly to wash remaining paraformaldehyde immobile liquid.The prescription of rinsing liquid is 0.38 gram glycocoll, 2.5 gram sucrose and 100 milliliters of 0.01MOL/L PBS solution.
Then, full brain sample being put into the pre-cooled concentration of frozen water successively is to carry out gradient dehydration, each 2 hours in 50%, 70% and 95% ethanolic solution.
After dehydration finished, it was to carry out gradient penetration, each 3 hours in 70%, 85%, 100% the GMA embedding liquid that full brain sample is put into concentration successively.Wherein the solvent of 70% and 85% GMA embedding liquid is 95% ethanol.Then put into fresh 100% GMA embedding liquid infiltration 12 hours once more, the GMA embedding liquid of putting into pre-polymerization at last permeated 3 days.All penetrating fluids all use ice-water bath pre-cooled earlier.In order to keep mouse brain YFP fluorescence, all add NaOH solution at the GMA of all concentration embedding liquid and regulate its PH to 9.5.The prescription of 100% GMA embedding liquid is: 67 gram GMA monomers, 3 gram water, 30 gram butyl methacrylates, 0.6 gram benzoyl peroxide.
The GMA embedding liquid making method of pre-polymerization is: getting 30 ml concns is that 100% GMA embedding liquid is put into 150 milliliters of conical flasks, seal the back with pan paper and bungee and insert a mercury thermometer, and the contact bottle end.Conical flask is put into heated and stirred on the heating magnetic stirring apparatus.When mercury thermometer indicated temperature to 120 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, reduce to the temperature of ice bath until solution temperature.
The full brain of mouse is put into gel capsule, and the GMA embedding liquid that adds pre-polymerization leaves standstill the bubble complete obiteration in capsule in about 10 minutes to the top, covers the capsule lid then.The full brain of mouse of capsule embedding vertically is placed on the capsule support, puts into 60 ℃ of baking oven polymerizations 60 hours then together, obtain required full brain embedding sample.
The full brain sample of gained is carried out continuous 1 micron slice and YFP fluorescence imaging, and gained section fluoroscopic image is with reference to Fig. 1.
Embodiment 2
After the Thy1-eYFP-H transgenic mice of growing up is anaesthetized with 1% yellow Jackets; 0.01MOL/L PBS solution through 37 ℃ of heart left ventricle perfusions 5 minutes; After treating that blood is rinsed well, pouring into the pre-cooled concentration of frozen water immediately is 4% paraformaldehyde immobile liquid, and continues 1 hour.Then, take out the full brain of mouse, be cut into the brain sheet of 1 millimeters thick, putting into the pre-cooled concentration of frozen water once more is fixing 6 hours of 4% paraformaldehyde immobile liquid back.Wherein the prescription of immobile liquid is 4 gram paraformaldehyde powder, 2.5 gram sucrose and 100 milliliters of 0.01MOL/L PBS solution.
Behind fixing the end, brain sheet sample is put into the pre-cooled rinsing liquid rinsing of frozen water 3 hours, more renew liquid 3 times therebetween, thoroughly to wash remaining paraformaldehyde immobile liquid.The prescription of rinsing liquid is 0.38 gram glycocoll, 2.5 gram sucrose and 100 milliliters of 0.01MOL/L PBS solution.
Then, brain sheet sample being put into the pre-cooled concentration of frozen water successively is to carry out gradient dehydration, each 15 minutes in 50%, 70% and 95% ethanolic solution.
After dehydration finished, it was to carry out gradient penetration, each 1 hour in 70%, 85%, 100% the GMA embedding liquid that brain sheet sample is put into concentration successively.Wherein concentration is that the solvent of 70% and 85% GMA embedding liquid is 95% ethanol.Then putting into fresh concentration once more is 100% GMA embedding liquid infiltration 6 hours, and the GMA embedding liquid of putting into pre-polymerization at last permeated 1 day.All penetrating fluids all use ice-water bath pre-cooled earlier.In order to keep mouse brain YFP fluorescence, all add NaOH solution at the GMA of all concentration embedding liquid and regulate its PH to 9.5.Concentration is that the prescription of 100% GMA embedding liquid is: 67 gram GMA monomers, 3 gram water, 30 gram butyl methacrylates, 0.6 gram benzoyl peroxide.
The GMA embedding liquid making method of pre-polymerization is: getting 30 ml concns is that 100% GMA embedding liquid is put into 150 milliliters of conical flasks, seal the back with pan paper and bungee and insert a mercury thermometer, and the contact bottle end.Conical flask is put into heated and stirred on the heating magnetic stirring apparatus.When mercury thermometer indicated temperature to 115 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, reduce to the temperature of ice bath until solution temperature.
The brain sheet is put into gel capsule, and the GMA embedding liquid that adds pre-polymerization leaves standstill the bubble complete obiteration in capsule in about 10 minutes to the top, covers the capsule lid then.The brain sheet of capsule embedding vertically is placed on the capsule support, put into 58 ℃ of baking oven polyase 13s then together 6 hours, obtain required brain sheet embedding sample.
Gained brain sheet sample carries out continuous 0.5 micron slice and fluorescence imaging, and the gained fluoroscopic image is with reference to Fig. 2.
Embodiment 3
After the GFP-M transgenic mice of being born back 20 days is anaesthetized with 1% yellow Jackets; 0.01MOL/L PBS solution through 37 ℃ of heart left ventricle perfusions 5 minutes; After treating that blood is rinsed well, pouring into the pre-cooled concentration of frozen water immediately is 4% paraformaldehyde immobile liquid, and continues 1 hour.Then, take out the full brain of mouse, putting into the pre-cooled concentration of frozen water once more is fixing 24 hours of 4% paraformaldehyde immobile liquid back.Wherein the prescription of immobile liquid is 4 gram paraformaldehyde powder, 2.5 gram sucrose and 100 milliliters of 0.01MOL/L PBS solution.
Behind fixing the end, full brain sample is put into the pre-cooled rinsing liquid rinsing of frozen water 12 hours, more renew liquid 3 times therebetween, thoroughly to wash remaining paraformaldehyde immobile liquid.The prescription of rinsing liquid is 0.38 gram glycocoll, 2.5 gram sucrose and 100 milliliters of 0.01MOL/L PBS solution.
Then, full brain sample being put into the pre-cooled concentration of frozen water successively is to carry out gradient dehydration, each 1 hour in 50%, 70% and 95% ethanolic solution.
After dehydration finished, it was to carry out gradient penetration, each 1.5 hours in 70%, 85%, 100% the GMA embedding liquid that full brain sample is put into concentration successively.Wherein concentration is that the solvent of 70% and 85% GMA embedding liquid is that concentration is 95% ethanol.Then put into fresh 100% GMA embedding liquid infiltration 12 hours once more, the GMA embedding liquid of putting into pre-polymerization at last permeated 2 days.All penetrating fluids all use ice-water bath pre-cooled earlier.In order to keep mouse brain YFP fluorescence, all add NaOH solution at the GMA of all concentration embedding liquid and regulate its PH to 8.The prescription of 100% GMA embedding liquid is: 67 gram GMA monomers, 3 gram water, 30 gram butyl methacrylates, 0.6 gram benzoyl peroxide.
The GMA embedding liquid making method of pre-polymerization is: getting 30 ml concns is that 100% GMA embedding liquid is put into 150 milliliters of conical flasks, seal the back with pan paper and bungee and insert a mercury thermometer, and the contact bottle end.Conical flask is put into heated and stirred on the heating magnetic stirring apparatus.When mercury thermometer indicated temperature to 117 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, reduce to the temperature of ice bath until solution temperature.
The full brain of mouse is put into gel capsule, and the GMA embedding liquid that adds pre-polymerization leaves standstill the bubble complete obiteration in capsule in about 10 minutes to the top, covers the capsule lid then.The full brain of mouse of capsule embedding vertically is placed on the capsule support, puts into 59 ℃ of baking oven polymerizations 48 hours then together, obtain required full brain embedding sample.
The full brain sample of gained carries out continuous 1 micron slice and fluorescence imaging, and the gained fluoroscopic image is with reference to Fig. 3.
It should be noted last that; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although with reference to preferred embodiment the present invention is specified, those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention; And not breaking away from the spirit and the scope of technical scheme of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (10)
1. a biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging is characterized in that, may further comprise the steps:
(1) biological sample being used the pre-cooled concentration of frozen water is fixing 6～24 hours of 4% paraformaldehyde immobile liquid;
(2) biological sample that step (1) is obtained cleaned 3～24 hours with the pre-cooled rinsing liquid of frozen water, changed fresh rinsing liquid 3 times therebetween;
(3) it is to carry out gradient dehydration, each 15 minutes to 2 hours in 50%, 70% and 95% ethanolic solution that the biological sample that step (2) is obtained is put into the pre-cooled concentration of frozen water successively;
(4) it is 70% that the biological sample that step (3) is obtained is put into the pre-cooled concentration of frozen water successively; Carry out gradient penetration in the water-soluble embedding liquid of 85% and 100% glycolmethacrylate (GMA); Each infiltration 1～3 hour; Put into fresh concentration subsequently once more and be 100% the water-soluble embedding liquid of GMA and permeated 6～12 hours, in the water-soluble embedding liquid of the GMA of pre-polymerization, permeated 1～3 day at last;
(5) biological sample that step (4) is obtained is put into the embedding mould oxygen barrier embedding of the water-soluble embedding liquid of the GMA that is full of pre-polymerization, inserts in 58 ℃～60 ℃ baking ovens polyase 13 then 6～60 hours.
2. the biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging according to claim 1 is characterized in that, the immobile liquid in the said step (1) is 4% paraformaldehyde powder by concentration, and 2.5% sucrose and 0.01 MOL/L PBS are formulated.
3. the biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging according to claim 1 is characterized in that, in the said step (1) if the back was fixing again after if the size of biological sample, was cut into the piece of tissue of 2 centimeter square earlier excessive.
4. the biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging according to claim 1 is characterized in that, rinsing liquid is that 0.38% glycocoll, 2.5% sucrose and 0.01MOL/L PBS are formulated by concentration in the said step (2).
5. the biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging according to claim 1; It is characterized in that; Said step (4) concentration is that 100% the water-soluble embedding liquid of GMA is by 67 gram GMA monomers; 3 gram water, 30 gram butyl methacrylates and 0.6 gram benzoyl peroxide are formulated.
6. the biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging according to claim 1; It is characterized in that; Be added with NaOH solution in the water-soluble embedding liquid of GMA in said step (4) and (5), the pH value that makes the water-soluble embedding liquid of GMA is 8～9.5.
7. the biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging according to claim 1; It is characterized in that; The water-soluble embedding liquid making method of the GMA of the pre-polymerization in said step (4) and (5) is: gets concentration and is 100% the water-soluble embedding liquid of GMA and put into conical flask, seal the back with pan paper and bungee and insert a mercury thermometer, and a contact bottle end; Conical flask is put into heated and stirred on the heating magnetic stirring apparatus; When mercury thermometer indicated temperature to 115 ℃～120 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, reduce to the temperature of ice bath until solution temperature.
8. the biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging according to claim 1; It is characterized in that; The embedding mould is the embedding mould with oxygen barrier function in the said step (5), and said embedding mould with oxygen barrier function can be gel capsule and BEEM capsule.
9. the biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging according to claim 1; It is characterized in that; In said (5) if there is not the embedding mould of oxygen barrier function; Embedding biological sample under oxygen free condition is put into airtight box with the embedding mould then, puts into the baking oven polymerization at last again.
10. according to each described biological sample preparation method who is applicable to ultra-thin section and fluorescence imaging of claim 1 to 9, it is characterized in that said biological sample can be the animal tissue of fluorescent protein labeling.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210092666.5A CN102620966B (en) | 2012-03-31 | 2012-03-31 | Biological sample preparation method applicable to ultra-thin section and fluorescence imaging |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210092666.5A CN102620966B (en) | 2012-03-31 | 2012-03-31 | Biological sample preparation method applicable to ultra-thin section and fluorescence imaging |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102620966A true CN102620966A (en) | 2012-08-01 |
| CN102620966B CN102620966B (en) | 2014-05-07 |
Family
ID=46561014
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210092666.5A Active CN102620966B (en) | 2012-03-31 | 2012-03-31 | Biological sample preparation method applicable to ultra-thin section and fluorescence imaging |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102620966B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102944456A (en) * | 2012-11-07 | 2013-02-27 | 西北农林科技大学 | Preparation method and application of tissue slice for observing temporal-spatial distribution of early embryo development in vivo |
| CN103471903A (en) * | 2013-09-29 | 2013-12-25 | 吴天骥 | Method for preparing stationary liquid for slicing and application of stationary liquid for slicing |
| CN103808553A (en) * | 2014-01-27 | 2014-05-21 | 华中科技大学 | Weak background fluorescence type resin embedding method |
| CN105021431A (en) * | 2014-04-24 | 2015-11-04 | 华中科技大学 | Resin embedding method for biological tissues marked by fluorescent protein and application of alkaline solution |
| CN106289915A (en) * | 2016-09-30 | 2017-01-04 | 中国科学院自动化研究所 | Water displacement composition, dewatering and preparation method for biological organization sample |
| CN106353160A (en) * | 2016-09-30 | 2017-01-25 | 中国科学院自动化研究所 | Protecting method for fluorescence signal of fluorescent protein in tissue sample and dehydration composition |
| CN106525792A (en) * | 2016-10-31 | 2017-03-22 | 华中科技大学 | Fluorescence control method of light-controlled fluorescent protein marker biological tissue embedding sample |
| CN112665951A (en) * | 2020-12-22 | 2021-04-16 | 中国农业科学院作物科学研究所 | Embryo milk tissue embedding method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1724652A (en) * | 2005-07-06 | 2006-01-25 | 清华大学 | Ectobody loaded with exogenous ligand molecule and its preparation method and application |
| CN1995350A (en) * | 2006-11-10 | 2007-07-11 | 西南大学 | Cotton fiber specific promoter and its use |
| CN101129396A (en) * | 2007-08-07 | 2008-02-27 | 遵义医学院 | Application of epimedium brevicornum glycosides in preparing medicament for treating senile dementia and product thereof |
-
2012
- 2012-03-31 CN CN201210092666.5A patent/CN102620966B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1724652A (en) * | 2005-07-06 | 2006-01-25 | 清华大学 | Ectobody loaded with exogenous ligand molecule and its preparation method and application |
| CN1995350A (en) * | 2006-11-10 | 2007-07-11 | 西南大学 | Cotton fiber specific promoter and its use |
| CN101129396A (en) * | 2007-08-07 | 2008-02-27 | 遵义医学院 | Application of epimedium brevicornum glycosides in preparing medicament for treating senile dementia and product thereof |
Non-Patent Citations (3)
| Title |
|---|
| 戴大临、张清敏: "《生物医学电镜样品制备方法》", 31 December 1993, article "包埋剂配方", pages: 62-63 * |
| 浦权等: "骨髓活检乙二醇甲基丙烯酸酯冷包埋切片免疫组织化学方法的建立", 《中华病理学杂志》, 28 February 1999 (1999-02-28) * |
| 邵淑娟: "《实用电子显微镜技术》", 31 December 2007, article "超薄切片制备程序", pages: 42-50 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102944456A (en) * | 2012-11-07 | 2013-02-27 | 西北农林科技大学 | Preparation method and application of tissue slice for observing temporal-spatial distribution of early embryo development in vivo |
| CN103471903A (en) * | 2013-09-29 | 2013-12-25 | 吴天骥 | Method for preparing stationary liquid for slicing and application of stationary liquid for slicing |
| CN103808553A (en) * | 2014-01-27 | 2014-05-21 | 华中科技大学 | Weak background fluorescence type resin embedding method |
| CN105021431A (en) * | 2014-04-24 | 2015-11-04 | 华中科技大学 | Resin embedding method for biological tissues marked by fluorescent protein and application of alkaline solution |
| CN106289915A (en) * | 2016-09-30 | 2017-01-04 | 中国科学院自动化研究所 | Water displacement composition, dewatering and preparation method for biological organization sample |
| CN106353160A (en) * | 2016-09-30 | 2017-01-25 | 中国科学院自动化研究所 | Protecting method for fluorescence signal of fluorescent protein in tissue sample and dehydration composition |
| CN106525792A (en) * | 2016-10-31 | 2017-03-22 | 华中科技大学 | Fluorescence control method of light-controlled fluorescent protein marker biological tissue embedding sample |
| CN112665951A (en) * | 2020-12-22 | 2021-04-16 | 中国农业科学院作物科学研究所 | Embryo milk tissue embedding method and application |
| CN112665951B (en) * | 2020-12-22 | 2022-11-11 | 中国农业科学院作物科学研究所 | Embryo milk tissue embedding method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102620966B (en) | 2014-05-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102620966B (en) | Biological sample preparation method applicable to ultra-thin section and fluorescence imaging | |
| CN102822657B (en) | Cell count and sample chamber and assembly method thereof | |
| Markert et al. | 3D subcellular localization with superresolution array tomography on ultrathin sections of various species | |
| Held et al. | Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy | |
| Susaki et al. | Perspective: extending the utility of three-dimensional organoids by tissue clearing technologies | |
| Yoshida et al. | Spheroid cultures of primary urothelial cancer cells: cancer tissue-originated spheroid (CTOS) method | |
| Sikandar | Histopathology: An old yet important technique in modern science | |
| US11365213B2 (en) | Composition for clearing spheroids, method for clearing spheroids using same, and kit comprising same | |
| Titze et al. | Ex vivo fluorescence confocal microscopy in specimens of the liver: a proof-of-concept study | |
| Phillips | Monitoring neuron and astrocyte interactions with a 3D cell culture system | |
| Eisenstein | TRANSPARENT TISSUES BRING BIOLOGY INTO FOCUS | |
| CN103808553B (en) | Weak background fluorescence type resin embedding method | |
| Goetz et al. | Using correlative light and electron microscopy to study zebrafish vascular morphogenesis | |
| Zhu et al. | Optimal combinations of fluorescent vessel labeling and tissue clearing methods for three-dimensional visualization of vasculature | |
| Wang et al. | FACT or PACT: A comparison between free-acrylamide and acrylamide-based passive sodium dodecyl sulfate tissue clearing for whole tissue imaging | |
| Bhattacharyya et al. | High-quality immunofluorescence of cultured cells | |
| Pawłowska et al. | Light-sheet microscopy for whole-brain imaging | |
| US20210231539A1 (en) | Composition for Immunostaining Cleared Large Tissues and Method for Immunostaining Cleared Large Biological Tissues | |
| Campinho et al. | Three-dimensional microscopy and image analysis methodology for mapping and quantification of nuclear positions in tissues with approximate cylindrical geometry | |
| Annese et al. | IKOSA® CAM Assay Application to Quantify Blood Vessels on Chick Chorioallantoic Membrane (CAM) | |
| KR102477568B1 (en) | A method of pretreatment of transparency of a biological samples with a size of 1 mm or less and a method of transparency of the biological samples comprising the same | |
| Ma et al. | Multiscale analysis of cellular composition and morphology in intact cerebral organoids | |
| Sutrisno et al. | Combined method of whole mount and block‐face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization | |
| Wang et al. | Three-dimensional autofocusing visual feedback for automated rare cells sorting in fluorescence microscopy | |
| Sumbal et al. | Single organoids droplet-based staining method for high-end 3D imaging of mammary organoids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |