CN102620966B - 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
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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 Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging.
Background technology
From the fluorescin nineties in 20th century (GFP) and derivant (XFPs) thereof, be found and be applied to zoology as a kind of novel markings thing, 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, XFPs and any interested protein can be coupled together, then by observing its fluorescence, thereby can observe the locus that is labeled albumen, motion and interaction.Moreover, by XFPs labelling technique, can also the various cells of specific marker, as three-dimensional spatial distribution and the annexation of neuron in brain, the 26S Proteasome Structure and Function relation of postgraduate's object is had to vital role.
In research in early days, generally adopt the biological sample imaging of common fluorescent microscope to XFPs mark, but owing to being subject to the restriction of Depth of field can only obtain the two dimensional image of slice sample.Afterwards, copolymerization application burnt and Two Photon Fluorescence technology has significantly increased the imaging depth to fluorescent samples, but owing to being subject to the restriction of the optical parametrics such as tissue scatter, its maximum imaging depth is still less than 1 millimeter, still cannot be for obtaining the three-dimensional data of cm size mcroorganism sample.Nearly ten years, in order to carry out high-precision three-dimensional imaging to large scale biological sample, development a series of novel optical imaging techniques that improve sample axial resolution and areas imaging by mechanical slice, as 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, are all the embedding method that adopts Electronic Speculum substantially, adopt resin embedding biological sample.
The resin using in Electronic Speculum generally can be divided into two classes, and a class is epikote, as Spurr and 812 etc., has hydrophobic property, needs sample to dewater completely before use; Another kind of is acrylic resin, and as LR White, LR Gold and Lowicryl etc., have water-wet behavior, allows that sample contains a certain amount of moisture before use.Have bibliographical information, the physico chemical factor that some are extreme, as high temperature, strong reductant-oxidant, strong acid and strong base and completely dehydration etc., all can cause the fluorescence intensity of XFPs to reduce even cancellation, so need epikote that sample dewaters not completely to be suitable for preparing the biological sample of XFPs mark.There is in recent years researcher to start to attempt with hydrophilic acryl resin, coming the biological sample of embedding XFPs mark under condition of ultralow temperature, as the cell of GFP or RFP mark, zebrafish embryo, nematode etc., gained sample can carry out ultra-thin section also can be for obtaining fluoroscopic image, as 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: (1) has been used the expensive device such as quick high-pressure frigorimeter and freezing substitution instrument, cannot promote in common lab; (2) quick high-pressure frigorimeter is only for the treatment of the tissue that is less than 200 micron thickness, so the biological sample that the sample preparation method based on this equipment also can only be less than 200 microns for the treatment of size; (3) used the heavy metal class immobile liquids such as acetic acid uranium or osmium tetroxide, not only there is very large toxicity, but also can reduce the fluorescence of XFPs; (4) still there is a certain amount of fluorescence losses in gained biological sample, have even up to more than 40%; (5) all operations all need to carry out under the ultra-low temperature surroundings of-90 ℃ to-20 ℃, and complex operation, only has the personnel through professional training to grasp.
In view of the novel three-dimensional formation methods such as microoptic computed tomography (SPECT) system (MOST) can carry out continuous ultra-thin section and imaging to the large-sized biological sample of centimetre-sized, and be the section real time imagery to motion in slicing processes, so require the fluorescence signal of sample higher, and existing fluorescent samples preparation method can not meet preparation large scale biological sample and the requirement that keeps fluorescence completely simultaneously.Therefore it is necessary, developing a kind of technique large scale Bio-specimen Preparation method simple, that be suitable for ultra-thin section and fluorescence imaging.
Summary of the invention
The object of the invention is for a kind of Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging that provides is provided.The 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 solution adopted in the present invention is:
A Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging, comprises the following steps:
(1) biological sample is fixed to 6～24 hours with the paraformaldehyde immobile liquid that the pre-cooled concentration of frozen water is 4%;
(2) biological sample step (1) being obtained cleans 3～24 hours with the pre-cooled rinsing liquid of frozen water, changes fresh rinsing liquid 3 times therebetween;
(3) it is to carry out gradient dehydration in 50%, 70% and 95% ethanolic solution that biological sample step (2) being obtained is put into the pre-cooled concentration of frozen water successively, each 15 minutes to 2 hours;
(4) it is 70% that biological sample step (3) being obtained is put into the pre-cooled concentration of frozen water successively, in the water-soluble embedding liquid of 85% and 100% glycolmethacrylate (GMA), carry out gradient penetration, each infiltration 1～3 hour, again put into subsequently fresh concentration and be 100% the water-soluble embedding liquid of GMA and permeate 6～12 hours, finally in the water-soluble embedding liquid of the GMA of pre-polymerization, permeate 1～3 day;
(5) biological sample step (4) being obtained is put into the imbedded mold oxygen barrier embedding of the water-soluble embedding liquid of GMA that is full of pre-polymerization, then inserts in 58 ℃～60 ℃ baking ovens polyase 13 6～60 hours.
Preferably, the immobile liquid in described step (1) is 4% paraformaldehyde powder by concentration, and 2.5% sucrose and 0.01MOL/L PBS are formulated.
Further, described step (1) if in the tissue of biological sample to anoxic sensitivity, can first pass through animal hearts perfusion fixation, then take out required tissue and put into the immobile liquid that the pre-cooled concentration of frozen water is 4% and be fixed.
Further, described step (1) if if in the size of biological sample excessive, be first cut into after the tissue block of 2 centimeter square fixing afterwards again.
Preferably, in described step (2), rinsing liquid is that 0.38% glycocoll, 2.5% sucrose and 0.01MOL/L PBS are formulated by concentration.
Preferably, the water-soluble embedding liquid of GMA that described step (4) concentration is 100% is by 67 grams of GMA monomers, 3 grams of water, and 30 grams of butyl methacrylates and 0.6 gram of benzoyl peroxide are formulated.
Further, in the water-soluble embedding liquid of GMA in described step (4) and (5), be added with NaOH solution, 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 acidity, can reduce the fluorescence of biological sample, in order to improve fluorescence intensity, so add NaOH solution, regulate its pH value.Add NaOH amount according to pH value corresponding to different fluorescin maximum fluorescence intensities, determine.
Preferably, the water-soluble embedding liquid making method of GMA of the pre-polymerization in described step (4) and (5) is: get concentration and be 100% the water-soluble embedding liquid of GMA and put into conical flask, with inserting a mercury thermometer after pan paper and bungee sealing, and the contact bottle end, conical flask is put on heating magnetic stirring apparatus and adds thermal agitation, when mercury thermometer indicated temperature to 115 ℃～120 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, until solution temperature is down to the temperature of ice bath.
Preferably, imbedded mold is the imbedded mold with oxygen barrier function in described step (5), described in there is oxygen barrier function imbedded mold can be gel capsule and BEEM capsule.
Further, described (5) if in there is no the imbedded mold of oxygen barrier function, also can then imbedded mold be put into airtight box at embedding biological sample under oxygen free condition, finally put into again baking oven polymerization.
Preferably, described biological sample can be the animal tissue of any fluorescent protein labeling.
The present invention has the following advantages:
(1) biological sample of the method energy embedding cm size; (2) the method can meet the requirement that sample hardness is applicable to continuous ultra-thin section; (3) the method can keep the fluorescence signal of sample completely; (4) this method step is simple, with low cost, the instrument of use, and the experiment conditions such as reagent and temperature are all the routine configurations of common lab, those skilled in the art can grasp.The biological sample of gained embedding, in conjunction with ultra-thin section 3 Dimension Image Technique, can be used for obtaining the meticulous three-dimensional fluorescence distribution of fluorescent protein labeling tissue, then carries out dependency structure and functional study.
Accompanying drawing explanation
Below in conjunction with drawings and embodiments, the present invention is further detailed explanation.
1 micron of section fluoroscopic image of the adult Thy1-eYFP-H Mouse Whole Brain sample that Fig. 1 is prepared for a kind of Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging that the embodiment of the present invention 1 provides.
0.5 micron of section fluoroscopic image of 1 millimeter of thickness brain sheet of adult Thy1-eYFP-H mouse that Fig. 2 is prepared for a kind of Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging that the embodiment of the present invention 2 provides.
1 micron of section fluoroscopic image of the full brain sample of GFP-M transgenic mice childhood that Fig. 3 is prepared for a kind of Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging that the embodiment of the present invention 3 provides.
Embodiment
Embodiment 1
Adult Thy1-eYFP-H transgenic mice is with after 1% yellow Jackets anesthesia, by heart left ventricle, pour into the 0.01MOL/L PBS solution 3 minutes of 37 ℃, after blood is rinsed well, pouring into immediately the pre-cooled concentration of frozen water is 4% paraformaldehyde immobile liquid, and continues 1 hour.Then, take out Mouse Whole Brain, again putting into the pre-cooled concentration of frozen water is to fix 24 hours after 4% paraformaldehyde immobile liquid.Wherein the formula of immobile liquid is 4 grams of paraformaldehyde powder, 2.5 grams of sucrose and 100 milliliters of 0.01MOL/L PBS solution.
After fixing 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 formula of rinsing liquid is 0.38 gram of glycocoll, 2.5 grams of sucrose and 100 milliliters of 0.01MOL/L PBS solution.
Then, it is to carry out gradient dehydration in 50%, 70% and 95% ethanolic solution that full brain sample is put into the pre-cooled concentration of frozen water successively, each 2 hours.
After dehydration finishes, it is to carry out gradient penetration, each 3 hours in 70%, 85%, 100% 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 again put into 100% fresh GMA embedding liquid and permeate 12 hours, the GMA embedding liquid of finally putting into pre-polymerization permeates 3 days.All penetrating fluids all first use ice-water bath pre-cooled.In order to keep mouse brain YFP fluorescence, at the GMA of all concentration embedding liquid, all add NaOH solution and regulate its PH to 9.5.The formula of 100% GMA embedding liquid is: 67 grams of GMA monomers, 3 grams of water, 30 grams of butyl methacrylates, 0.6 gram of 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, with inserting a mercury thermometer after pan paper and bungee sealing, and the contact bottle end.Conical flask is put on heating magnetic stirring apparatus and adds thermal agitation.When mercury thermometer indicated temperature to 120 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, until solution temperature is down to the temperature of ice bath.
Mouse Whole Brain is put into gel capsule, add the GMA embedding liquid of pre-polymerization to top, standing approximately 10 minutes until the bubble in capsule disappears completely, then covers capsule lid.The Mouse Whole Brain of capsule embedding is vertically placed on capsule support, then puts into together 60 ℃ of baking oven polymerizations 60 hours, obtain required full brain embedding sample.
The full brain sample of gained is carried out to continuous 1 micron of slice and YFP fluorescence imaging, and gained section fluoroscopic image is with reference to Fig. 1.
Embodiment 2
Grow up Thy1-eYFP-H transgenic mice with after 1% yellow Jackets anesthesia, by heart left ventricle, pour into the 0.01MOL/L PBS solution 5 minutes of 37 ℃, after blood is rinsed well, pouring into immediately the pre-cooled concentration of frozen water is 4% paraformaldehyde immobile liquid, and continues 1 hour.Then, take out Mouse Whole Brain, be cut into the brain sheet of 1 millimeters thick, again putting into the pre-cooled concentration of frozen water is to fix 6 hours after 4% paraformaldehyde immobile liquid.Wherein the formula of immobile liquid is 4 grams of paraformaldehyde powder, 2.5 grams of sucrose and 100 milliliters of 0.01MOL/L PBS solution.
After fixing 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 formula of rinsing liquid is 0.38 gram of glycocoll, 2.5 grams of sucrose and 100 milliliters of 0.01MOL/L PBS solution.
Then, it is to carry out gradient dehydration in 50%, 70% and 95% ethanolic solution that brain sheet sample is put into the pre-cooled concentration of frozen water successively, each 15 minutes.
After dehydration finishes, it is to carry out gradient penetration, each 1 hour in 70%, 85%, 100% 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 again putting into fresh concentration is that 100% GMA embedding liquid permeates 6 hours, and the GMA embedding liquid of finally putting into pre-polymerization permeates 1 day.All penetrating fluids all first use ice-water bath pre-cooled.In order to keep mouse brain YFP fluorescence, at the GMA of all concentration embedding liquid, all add NaOH solution and regulate its PH to 9.5.Concentration is that the formula of 100% GMA embedding liquid is: 67 grams of GMA monomers, 3 grams of water, 30 grams of butyl methacrylates, 0.6 gram of 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, with inserting a mercury thermometer after pan paper and bungee sealing, and the contact bottle end.Conical flask is put on heating magnetic stirring apparatus and adds thermal agitation.When mercury thermometer indicated temperature to 115 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, until solution temperature is down to the temperature of ice bath.
Brain sheet is put into gel capsule, add the GMA embedding liquid of pre-polymerization to top, standing approximately 10 minutes until the bubble in capsule disappears completely, then covers capsule lid.The brain sheet of capsule embedding is vertically placed on capsule support, then puts into together 58 ℃ of baking oven polyase 13s 6 hours, obtain required brain sheet embedding sample.
Gained brain sheet sample carries out continuous 0.5 micron of slice and fluorescence imaging, and gained fluoroscopic image is with reference to Fig. 2.
Embodiment 3
The GFP-M transgenic mice of being born latter 20 days is with after 1% yellow Jackets anesthesia, by heart left ventricle, pour into the 0.01MOL/L PBS solution 5 minutes of 37 ℃, after blood is rinsed well, pouring into immediately the pre-cooled concentration of frozen water is 4% paraformaldehyde immobile liquid, and continues 1 hour.Then, take out Mouse Whole Brain, again putting into the pre-cooled concentration of frozen water is to fix 24 hours after 4% paraformaldehyde immobile liquid.Wherein the formula of immobile liquid is 4 grams of paraformaldehyde powder, 2.5 grams of sucrose and 100 milliliters of 0.01MOL/L PBS solution.
After fixing 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 formula of rinsing liquid is 0.38 gram of glycocoll, 2.5 grams of sucrose and 100 milliliters of 0.01MOL/L PBS solution.
Then, it is to carry out gradient dehydration in 50%, 70% and 95% ethanolic solution that full brain sample is put into the pre-cooled concentration of frozen water successively, each 1 hour.
After dehydration finishes, it is to carry out gradient penetration, each 1.5 hours in 70%, 85%, 100% 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 again put into 100% fresh GMA embedding liquid and permeate 12 hours, the GMA embedding liquid of finally putting into pre-polymerization permeates 2 days.All penetrating fluids all first use ice-water bath pre-cooled.In order to keep mouse brain YFP fluorescence, at the GMA of all concentration embedding liquid, all add NaOH solution and regulate its PH to 8.The formula of 100% GMA embedding liquid is: 67 grams of GMA monomers, 3 grams of water, 30 grams of butyl methacrylates, 0.6 gram of 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, with inserting a mercury thermometer after pan paper and bungee sealing, and the contact bottle end.Conical flask is put on heating magnetic stirring apparatus and adds thermal agitation.When mercury thermometer indicated temperature to 117 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, until solution temperature is down to the temperature of ice bath.
Mouse Whole Brain is put into gel capsule, add the GMA embedding liquid of pre-polymerization to top, standing approximately 10 minutes until the bubble in capsule disappears completely, then covers capsule lid.The Mouse Whole Brain of capsule embedding is vertically placed on capsule support, then puts into together 59 ℃ of baking oven polymerizations 48 hours, obtain required full brain embedding sample.
The full brain sample of gained carries out continuous 1 micron of slice and fluorescence imaging, and 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 the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (10)
1. a Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging, is characterized in that, comprises the following steps:
(1) biological sample is fixed to 6～24 hours with the paraformaldehyde immobile liquid that the pre-cooled concentration of frozen water is 4%;
(2) biological sample step (1) being obtained cleans 3～24 hours with the pre-cooled rinsing liquid of frozen water, changes fresh rinsing liquid 3 times therebetween;
(3) it is to carry out gradient dehydration in 50%, 70% and 95% ethanolic solution that biological sample step (2) being obtained is put into the pre-cooled concentration of frozen water successively, each 15 minutes to 2 hours;
(4) it is 70% that biological sample step (3) being obtained is put into the pre-cooled concentration of frozen water successively, in the water-soluble embedding liquid of 85% and 100% glycolmethacrylate (GMA), carry out gradient penetration, each infiltration 1～3 hour, again put into subsequently fresh concentration and be 100% the water-soluble embedding liquid of GMA and permeate 6～12 hours, finally in the water-soluble embedding liquid of the GMA of pre-polymerization, permeate 1～3 day;
(5) biological sample step (4) being obtained is put into the imbedded mold oxygen barrier embedding of the water-soluble embedding liquid of GMA that is full of pre-polymerization, then inserts in 58 ℃～60 ℃ baking ovens polyase 13 6～60 hours.
2. the Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging according to claim 1, it is characterized in that, immobile liquid in described step (1) is 4% paraformaldehyde powder by concentration, and 2.5% sucrose and 0.01 MOL/L PBS are formulated.
3. the Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging according to claim 1, is characterized in that, described step (1) if if in the size of biological sample excessive, be first cut into after the tissue block of 2 centimeter square fixing afterwards again.
4. the Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging according to claim 1, is characterized in that, in described step (2), rinsing liquid is that 0.38% glycocoll, 2.5% sucrose and 0.01MOL/L PBS are formulated by concentration.
5. the Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging according to claim 1, it is characterized in that, the water-soluble embedding liquid of GMA that described step (4) concentration is 100% is by 67 grams of GMA monomers, 3 grams of water, 30 grams of butyl methacrylates and 0.6 gram of benzoyl peroxide are formulated.
6. the Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging according to claim 1, it is characterized in that, in the water-soluble embedding liquid of GMA in described step (4) and (5), be added with NaOH solution, the pH value that makes the water-soluble embedding liquid of GMA is 8～9.5.
7. the Bio-specimen Preparation method that 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 GMA of the pre-polymerization in described step (4) and (5) is: get concentration and be 100% the water-soluble embedding liquid of GMA and put into conical flask, with inserting a mercury thermometer after pan paper and bungee sealing, and the contact bottle end, conical flask is put on heating magnetic stirring apparatus and adds thermal agitation, when mercury thermometer indicated temperature to 115 ℃～120 ℃, rapidly conical flask is put into ice-water bath, and shake energetically, until solution temperature is down to the temperature of ice bath.
8. the Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging according to claim 1, it is characterized in that, imbedded mold is the imbedded mold with oxygen barrier function in described step (5), described in there is oxygen barrier function imbedded mold can be gel capsule and BEEM capsule.
9. the Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging according to claim 1, it is characterized in that, described (5) if in there is no the imbedded mold of oxygen barrier function, embedding biological sample under oxygen free condition, then imbedded mold is put into airtight box, finally put into again baking oven polymerization.
10. according to the Bio-specimen Preparation method that is applicable to ultra-thin section and fluorescence imaging described in claim 1 to 9 any one, it is characterized in that, described biological sample can be the animal tissue of fluorescent protein labeling.
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| 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 |
| CN103471903B (en) * | 2013-09-29 | 2016-01-06 | 吴天骥 | The collocation method of section immobile liquid and application thereof |
| CN103808553B (en) * | 2014-01-27 | 2017-04-19 | 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 |
| 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 |
| 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 |
| CN106525792B (en) * | 2016-10-31 | 2019-07-09 | 华中科技大学 | A kind of fluorescence control method of light-operated fluorescent protein labeling biological tissue embedding sample |
| 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 |
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