CN111122267A - Method for making semi-thin slice of plant tissue - Google Patents
Method for making semi-thin slice of plant tissue Download PDFInfo
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- CN111122267A CN111122267A CN201911355631.4A CN201911355631A CN111122267A CN 111122267 A CN111122267 A CN 111122267A CN 201911355631 A CN201911355631 A CN 201911355631A CN 111122267 A CN111122267 A CN 111122267A
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- dyeing
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000004043 dyeing Methods 0.000 claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 claims abstract description 19
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000011347 resin Substances 0.000 claims abstract description 13
- 229920005989 resin Polymers 0.000 claims abstract description 13
- 238000010186 staining Methods 0.000 claims abstract description 10
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 claims abstract description 6
- 229910000831 Steel Inorganic materials 0.000 claims abstract description 6
- 239000010959 steel Substances 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 70
- 241000196324 Embryophyta Species 0.000 claims description 15
- 230000018044 dehydration Effects 0.000 claims description 11
- 238000006297 dehydration reaction Methods 0.000 claims description 11
- 240000006394 Sorghum bicolor Species 0.000 claims description 10
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 6
- 238000001764 infiltration Methods 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 230000008595 infiltration Effects 0.000 claims description 5
- 230000000149 penetrating effect Effects 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 abstract description 12
- 229950003937 tolonium Drugs 0.000 abstract description 7
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 abstract description 7
- 239000003086 colorant Substances 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 239000012188 paraffin wax Substances 0.000 abstract description 4
- 239000001045 blue dye Substances 0.000 abstract description 2
- 239000002244 precipitate Substances 0.000 abstract description 2
- 238000003860 storage Methods 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 22
- 239000000243 solution Substances 0.000 description 11
- 239000007864 aqueous solution Substances 0.000 description 7
- 244000153158 Ammi visnaga Species 0.000 description 4
- 235000010585 Ammi visnaga Nutrition 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 3
- 239000012466 permeate Substances 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- KVNRLNFWIYMESJ-UHFFFAOYSA-N butyronitrile Chemical compound CCCC#N KVNRLNFWIYMESJ-UHFFFAOYSA-N 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 238000012137 double-staining Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 210000004243 sweat Anatomy 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 244000124209 Crocus sativus Species 0.000 description 1
- 235000015655 Crocus sativus Nutrition 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 239000001008 quinone-imine dye Substances 0.000 description 1
- 235000013974 saffron Nutrition 0.000 description 1
- 239000004248 saffron Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000979 synthetic dye Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a method for manufacturing a semi-thin slice of plant tissue. The manufacturing method of the plant tissue semi-thin slice comprises the following steps: selecting plant tissues, and sequentially fixing, dehydrating, pre-permeating, embedding, slicing, baking, removing resin and dyeing; slicing by using a steel blade; the staining was performed with safranin and fast green in sequence. The invention uses the steel blade commonly used in paraffin section as the section cutter, and enlarges the application range of resin section. Compared with toluidine blue dye liquor, the method for dyeing by using safranin-fast green counterdyeing has the advantages that the price of safranin is lower than that of toluidine blue, the storage requirement is not high, the safranin can be stored at room temperature, the preparation process is simple and easy to implement, precipitates are not easy to generate, different tissue structures show different colors, different structures and components of tissues can be more obviously embodied, and the quality of sections and the image effect are improved.
Description
Technical Field
The invention relates to a method for manufacturing a semi-thin slice of plant tissue, belonging to the technical field of biology.
Background
The resin semi-thin section is a morphology research technology with wide application, and is commonly used for positioning ultrathin section tissues in the preparation of transmission electron microscope samples. Compared with paraffin sections, the manufacturing procedure and steps of the resin semi-thin sections are simplified, the period is short, the thickness of the sections is thinner, the general thickness is only 2-7 mu m, therefore, the definition and the resolution of the semi-thin section images are superior to those of the paraffin sections, the visual field is larger than that of the ultra-thin sections, and the high-quality optical lens images are obtained. The steps from material taking to dyeing are involved, and the sample preparation quality is affected and even the sample preparation fails due to the problems in any link. The advantages and the characteristics of the resin semi-thin section technology can be reflected more perfectly by a good dyeing method. Toluidine blue is a commonly used artificial synthetic dye, a commonly used coloring agent in the preparation process of the plant semi-thin section belongs to quinone imine dyes, cations in the dyes have a dyeing effect, and acidic substances of tissue cells are combined with the cations in the dyes, so that the dyes are single dyes, lack of gradation and have no contrast, and meanwhile, a toluidine blue coloring agent solution is easy to precipitate and denature, which can influence the quality and the image effect of the semi-thin section. Therefore, in order to overcome the defects of the prior art, such as toluidine blue staining and high price of a glass cutter, a new method for manufacturing the semi-thin slice of the plant tissue needs to be provided.
Disclosure of Invention
The invention aims to provide a method for manufacturing a semi-thin slice of plant tissue.
The invention provides a method for manufacturing a semi-thin slice of plant tissue, which comprises the following steps:
selecting plant tissues, and sequentially fixing, dehydrating, pre-permeating, embedding, slicing, baking, removing resin and dyeing;
slicing by using a steel blade;
the staining was performed with safranin and fast green in sequence.
In the above preparation method, the fixing step can be performed by conventional method, such as in 2.5% glutaraldehyde fixing solution for more than 24h, and vacuum pumping is performed to fully fix tissues and cells; then rinsing the fixed tissue (such as leaf) with 0.1M PBS buffer solution for 3 times, each time for 20 min; rinsing with distilled water for 20min for 2 times, and washing off the fixative completely.
In the above manufacturing method, the dehydration step is as follows: dewatering step by adopting 50-100% ethanol water solution, dewatering once by adopting 50-95% ethanol water solution, and placing for 1h in each gradient; dehydrating with 100% ethanol water solution twice, and standing for 1 hr each time;
if 50%, 70%, 80%, 90%, 95% and 100% ethanol aqueous solution are used for dehydration step by step, 50% ethanol aqueous solution can be used for dehydration for 1h, 70% ethanol aqueous solution can be used for dehydration for 1h, 80% ethanol aqueous solution can be used for dehydration for 1h, 90% ethanol aqueous solution can be used for dehydration for 1h, 95% ethanol aqueous solution can be used for dehydration for 1h, and 100% ethanol aqueous solution can be used for dehydration twice, 1h each time, in order to prevent over dehydration in pure ethanol, so that the pure ethanol can not be placed for more than 2 h.
In the above manufacturing method, the pre-permeation liquid used for the pre-permeation is prepared from a mixture of 1: 1 Technovit and ethanol;
the penetrating fluid consists of Technovit and Hardender I, and the mixture ratio of the Technovit to the Hardender I is 100 mlTechnovit: 1g of Hardender I;
the pre-infiltration time is 2.5-4 hours, and the infiltration time is 20-24 hours.
In the manufacturing method, the thickness of the slice obtained by slicing is 3-7 μm; in the slicing process, rubber or butyronitrile gloves are worn in the whole process, so that the embedded blocks are prevented from being softened by sweat on hands, and powder is easily formed in the slicing process. If curling occurs during the slicing process, the tip of the embedded block is too large, so that the smaller the tip, the better the tip. And cutting a slice in the slicing process, clamping the edge of the slice by using a pointed forceps, placing the slice on a glass slide on which distilled water is dripped, and slowly flattening the slice by using a toothpick.
In the above preparation method, the baking sheet can be carried out under conventional conditions; because the section is too thin, the section is slowly placed on a glass slide on which water drops are dropped by a toothpick, and after the tissue structure is preliminarily observed under a microscope to be complete, the section is placed on an electric hot plate at 60 ℃ for baking.
In the above manufacturing method, the resin removal can be performed under conventional conditions; and placing the observed and available slices in a staining jar filled with absolute ethyl alcohol for standing for 5min, and performing slice resin removal treatment.
In the preparation method, the safranin is adopted for standing and dyeing for 12-16 hours;
after the safranine dyeing, the method also comprises the step of cleaning and decoloring for 3-5 times by adopting water;
standing and dyeing for 40-60 min by adopting the green fixing;
after the safranine dyeing, the method also comprises the step of cleaning and decoloring for 4-6 times by adopting water;
the plant tissue to which the method of the present invention is applicable may be leaves, roots or stalks.
The plant is preferably sorghum.
The invention has the following beneficial effects:
(1) the invention uses the steel blade commonly used in paraffin section as the section cutter, and enlarges the application range of resin section.
(2) Compared with toluidine blue dye liquor, the method for dyeing by using safranin-fast green counterstaining has the advantages that the price of safranin is lower than that of toluidine blue, the storage requirement is not high, the safranin can be stored at room temperature, the preparation process is simple and easy to implement, precipitates are not easy to generate, different tissue structures show different colors, different structures and components of tissues can be more obviously embodied, and the quality of sections and the image effect are improved.
(3) The method of the invention can be applied to leaf tissue of sorghum, can be further applied to leaf, root and stem tissues of other plants, and plays an important role in application observation of plant tissue slices.
Drawings
FIG. 1 is a process of double staining of saffron with fixed green and semi-thin slices of sorghum leaves according to the present invention.
FIG. 2 is a photomicrograph of a semi-thin section (. times.20) of sorghum leaves.
FIG. 3 is a photomicrograph of a semi-thin section (. times.20) of sorghum radicle.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
As shown in FIG. 1, it is a flow chart of the present invention for double staining of sorghum leaf safranin green-fixing half thin slices:
(1) material taking: selecting leaves with size of 0.6cm × 0.6cm at different growth stages to remove vein;
(2) fixing: fixing the leaves in 2.5% glutaraldehyde fixing solution for over 24 hr, and vacuumizing to fix tissue and cell;
(3) rinsing: rinsing the fixed leaves with 0.1M PBS buffer solution for 3 times, each time for 20 min; rinsing with distilled water for 20min for 2 times, and washing to remove fixative;
(4) and (3) dehydrating: the ethanol is dehydrated step by 50%, 70%, 80%, 90%, 95% and 100% ethanol, 50% ethanol is dehydrated for 1h, 70% ethanol is dehydrated for 1h, 80% ethanol is dehydrated for 1h, 90% ethanol is dehydrated for 1h, 95% ethanol is dehydrated for 1h, 100% ethanol is dehydrated twice, and each time for 1h, in order to prevent excessive dehydration in pure ethanol, the ethanol cannot be placed in pure ethanol for more than 2 h.
(5) Configuration of the pre-permeate and permeate: the composition of the pre-permeation solution is 1/2Technovit + 1/2100% ethanol; the penetrating fluid consists of 100mL of Technit +1g of Hardender I, and can be placed at 4 ℃ for 4 weeks after being prepared. If the sample preparation hardness is not enough, the dosage of Hardender I can be increased in the process of preparing the penetrating fluid.
(6) Pre-infiltration: the dehydrated leaves were left to stand in the pre-permeation solution at room temperature for 3 hours.
(7) And (3) infiltration: the pre-infiltrated material was transferred to the permeate and allowed to infiltrate for 24 hours.
(8) Preparation of embedding liquid: the embedding solution consists of 1.5mL +0.1mL Hardender II and must be prepared for use. If the sample preparation hardness is not enough, the dosage of Hardender II can be increased in a proper amount in the process of preparing the penetrating fluid.
(9) Embedding: adding the freshly prepared embedding solution into an embedding plate with 21 holes, and adding 300 mu L of the embedding solution into each hole; placing the penetrated leaf tissue into a sample hole by using a pointed-end forceps, adjusting the direction and position of the sample by using a toothpick, immediately transferring into an oven, and curing for more than 5 days at the temperature of 45 ℃.
(10) Block repairing: the solidified embedded block was trimmed with 2000-mesh sandpaper to expose the tissue, observed under a stereomicroscope, and trimmed to have a tapered side surface.
(11) Semi-thin slicing: the trimmed embedded blocks were cut into semi-thin sections with a LEICA RM2265 microtome, and the steel blade was selected as the slicing knife, and the thickness of the sections was 4 μm. The butyronitrile gloves are worn in the whole operation process to prevent sweat on hands from softening the embedded blocks, so that powder is easily formed in the slicing process. If curling occurs during the slicing process, the tip of the embedded block is too large, so that the smaller the tip, the better the tip.
(12) Baking slices: because the section is too thin, the section is slowly placed on a glass slide on which water drops are dropped by a toothpick, and after the tissue structure is preliminarily observed under a microscope to be complete, the section is placed on an electric hot plate at 60 ℃ for baking.
(13) Resin removal: and placing the observed and available slices in a staining jar filled with absolute ethyl alcohol for standing for 5min, and performing slice resin removal treatment.
(14) Dyeing: transferring the slices subjected to resin removal to a staining jar filled with safranin, standing and staining for 15.5h, and after staining, placing the slices in a staining jar filled with ultrapure water to clean and decolorize for 3 times, wherein each time lasts for 1.5 min; and (3) placing the safranine dyed and cleaned slice into a dyeing tank filled with fast green for standing and dyeing for 50min, and placing the fast green dyed slice into a dyeing tank filled with ultrapure water for cleaning and decoloring for 5 times, wherein each time lasts for 2.0 min. The stained sections can be observed under a microscope.
Microscopic photographs of the half thin slices (. times.20) of sorghum leaves and the half thin slices (. times.20) of sorghum radicles obtained according to the above method are shown in FIGS. 1 and 2, respectively, and it can be seen that the tissue structures of the sorghum leaves and roots are clear.
Claims (8)
1. A method for manufacturing a semi-thin slice of plant tissue comprises the following steps:
selecting plant tissues, and sequentially fixing, dehydrating, pre-permeating, embedding, slicing, baking, removing resin and dyeing;
slicing by using a steel blade;
the staining was performed with safranin and fast green in sequence.
2. The method of manufacturing according to claim 1, wherein: the thickness of the slice obtained by slicing is 3-7 mu m.
3. The manufacturing method according to claim 1 or 2, characterized in that: the dyeing time of the safranin is 12-16 h;
and after the safranine dyeing, the method also comprises the step of cleaning and decoloring for 3-5 times by adopting water.
4. The production method according to any one of claims 1 to 3, characterized in that: the time for dyeing by adopting the fast green is 40-60 min;
and after the safranine dyeing, the method also comprises the step of cleaning and decoloring for 4-6 times by adopting water.
5. The production method according to any one of claims 1 to 4, wherein: the dehydration steps are as follows: dewatering step by adopting 50-100% ethanol water solution, dewatering once by adopting 50-95% ethanol water solution, and placing for 1h in each gradient; dehydrating with 100% ethanol water solution twice, and standing for 1 hr each time.
6. The production method according to any one of claims 1 to 5, wherein: the pre-permeation liquid adopted by the pre-permeation is prepared from the following components in percentage by volume of 1: 1 Technovit and ethanol;
the penetrating fluid consists of Technovit and Hardender I, and the mixture ratio of the Technovit to the Hardender I is 100 mlTechnovit: 1g of Hardender I;
the pre-infiltration time is 2.5-4 hours, and the infiltration time is 20-24 hours.
7. The production method according to any one of claims 1 to 6, characterized in that: the plant tissue is leaves, roots or stalks.
8. The production method according to any one of claims 1 to 7, characterized in that: the plant is sorghum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911355631.4A CN111122267A (en) | 2019-12-25 | 2019-12-25 | Method for making semi-thin slice of plant tissue |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911355631.4A CN111122267A (en) | 2019-12-25 | 2019-12-25 | Method for making semi-thin slice of plant tissue |
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| CN111122267A true CN111122267A (en) | 2020-05-08 |
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Citations (4)
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| CN103852368A (en) * | 2013-09-04 | 2014-06-11 | 南京农业大学 | Mitochondria DNA observation through MTG-DAPI double-staining of semi-thin sections of cucumber pollen |
| CN104390834A (en) * | 2014-11-25 | 2015-03-04 | 扬州大学 | Sarranine and methyl violet mixed staining method for resin slices and staining solution thereof |
| CN106546473A (en) * | 2016-11-26 | 2017-03-29 | 中国热带农业科学院南亚热带作物研究所 | It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud |
| CN109855936A (en) * | 2016-06-29 | 2019-06-07 | 西南大学 | A kind of preparation method of complete, conducive to microexamination the plant tissue slice of tissue |
-
2019
- 2019-12-25 CN CN201911355631.4A patent/CN111122267A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103852368A (en) * | 2013-09-04 | 2014-06-11 | 南京农业大学 | Mitochondria DNA observation through MTG-DAPI double-staining of semi-thin sections of cucumber pollen |
| CN104390834A (en) * | 2014-11-25 | 2015-03-04 | 扬州大学 | Sarranine and methyl violet mixed staining method for resin slices and staining solution thereof |
| CN109855936A (en) * | 2016-06-29 | 2019-06-07 | 西南大学 | A kind of preparation method of complete, conducive to microexamination the plant tissue slice of tissue |
| CN106546473A (en) * | 2016-11-26 | 2017-03-29 | 中国热带农业科学院南亚热带作物研究所 | It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud |
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