CN114813288B - Quick dyeing and positioning method for active oxygen of plant root system - Google Patents
Quick dyeing and positioning method for active oxygen of plant root system Download PDFInfo
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- 238000004043 dyeing Methods 0.000 title claims abstract description 61
- 241000196324 Embryophyta Species 0.000 title claims abstract description 48
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 44
- 239000001301 oxygen Substances 0.000 title claims abstract description 44
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 37
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000000243 solution Substances 0.000 claims abstract description 32
- 238000010186 staining Methods 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 239000012192 staining solution Substances 0.000 claims abstract description 20
- 238000007493 shaping process Methods 0.000 claims abstract description 16
- 239000007864 aqueous solution Substances 0.000 claims abstract description 13
- 238000007789 sealing Methods 0.000 claims abstract description 13
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 22
- 235000017784 Mespilus germanica Nutrition 0.000 claims description 17
- 244000182216 Mimusops elengi Species 0.000 claims description 17
- 235000000560 Mimusops elengi Nutrition 0.000 claims description 17
- 235000007837 Vangueria infausta Nutrition 0.000 claims description 17
- 239000011259 mixed solution Substances 0.000 claims description 12
- 230000007935 neutral effect Effects 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- YHVQIDWAIRCSOQ-UHFFFAOYSA-N 1-nitrotetrazol-2-ium chloride Chemical compound [Cl-].[O-][N+](=O)N1C=[NH+]N=N1 YHVQIDWAIRCSOQ-UHFFFAOYSA-N 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical compound C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 claims description 3
- DVLFYONBTKHTER-UHFFFAOYSA-M 3-(N-morpholino)propanesulfonate Chemical compound [O-]S(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-M 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 239000000839 emulsion Substances 0.000 claims description 2
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- YPKOTWSAVCIFAM-UHFFFAOYSA-N [Na].CCC Chemical compound [Na].CCC YPKOTWSAVCIFAM-UHFFFAOYSA-N 0.000 claims 1
- 238000004140 cleaning Methods 0.000 claims 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims 1
- 230000008557 oxygen metabolism Effects 0.000 abstract description 3
- CMDGQTVYVAKDNA-UHFFFAOYSA-N propane-1,2,3-triol;hydrate Chemical compound O.OCC(O)CO CMDGQTVYVAKDNA-UHFFFAOYSA-N 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000003292 glue Substances 0.000 description 16
- 241000223221 Fusarium oxysporum Species 0.000 description 14
- 239000011521 glass Substances 0.000 description 12
- 239000000463 material Substances 0.000 description 11
- 238000001514 detection method Methods 0.000 description 9
- 244000241838 Lycium barbarum Species 0.000 description 8
- 235000015459 Lycium barbarum Nutrition 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 238000001816 cooling Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000001744 histochemical effect Effects 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 238000009966 trimming Methods 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- -1 diphenylene iodide chloride Chemical compound 0.000 description 4
- 238000007598 dipping method Methods 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 239000003642 reactive oxygen metabolite Substances 0.000 description 4
- 238000010008 shearing Methods 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 238000005086 pumping Methods 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 235000015468 Lycium chinense Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- MQVMJSWYKLYFIG-UHFFFAOYSA-N propane-1-sulfonic acid;sodium Chemical compound [Na].CCCS(O)(=O)=O MQVMJSWYKLYFIG-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- NPAWNPCNZAPTKA-UHFFFAOYSA-M sodium;propane-1-sulfonate Chemical compound [Na+].CCCS([O-])(=O)=O NPAWNPCNZAPTKA-UHFFFAOYSA-M 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- 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
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
-
- 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
- G01N1/36—Embedding or analogous mounting of samples
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a rapid dyeing and positioning method for active oxygen of a plant root system, which comprises the following steps: (1) Fixing a plant root system to the bottom of a container, adding a staining solution corresponding to active oxygen to be detected, which is used for immersing a sample, and placing the sample into a vacuum container for low-temperature vacuum staining; (2) Discarding the dyeing liquid, adding the fixing liquid, and putting the dyeing liquid into a vacuum container for low-temperature vacuum fixing; (3) Discarding the fixing solution, adding the glycerol aqueous solution, and placing the mixture into a vacuum container for low-temperature vacuum filling and shaping; (4) Precooling the cold chamber and a slicing cutter, embedding plant root systems of which the glycerol water solution is discarded by using an OTC embedding agent, adjusting the temperature of a fixed table, and slicing; (5) sealing the plant root after the temperature is returned. The method saves a large amount of manufacturing time and realizes rapid slicing; the influence of temperature on active oxygen metabolism is reduced, and the slicing accuracy is greatly improved; can ensure that tissues can not shrink, keep the original life form and ensure the reality and reliability of slicing.
Description
Technical Field
The invention relates to a rapid dyeing and positioning method for active oxygen of a plant root system.
Background
Active oxygen metabolism in plants is very rapid, O 2 - Metabolism time is calculated in milliseconds, with the slowest metabolism of H 2 O 2 The time remaining in the body is not too long as a minute. The instability of the plant tissue needs to be considered when the plant tissue is dyed and positioned by active oxygen, and the higher the temperature is, the longer the treatment time is, the more unstable the plant tissue is, and the more inaccurate the result is.
The plant root system is cylindrical, and the method of reactive oxygen species staining and transparence of the plant leaf cannot be referred to, and the plant root system can be observed after being cut into slices. The traditional paraffin section needs to be subjected to steps of fixing, embedding, slicing, dyeing and the like, so that the time is long, the temperature of molten paraffin is high, the loss of active oxygen is excessive, and the paraffin section cannot be used for researching the active oxygen positioning.
Therefore, a rapid and accurate method must be selected for the localization study of reactive oxygen species.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a rapid dyeing and positioning method for active oxygen of a plant root system. The invention uses the low-temperature vacuum dyeing fixing and frozen slicing method to shorten the time length of active oxygen slicing from 72 hours to 12 hours, effectively improves the working efficiency, can greatly preserve the original state of active oxygen and improves the research accuracy. The method can be used for the rapid reactive oxygen species staining section observation of the plant root system with the diameter of 3-8 mm.
The invention provides a rapid dyeing and positioning method for active oxygen of a plant root system, which comprises the following steps:
(1) Fixing a plant root system to the bottom of a container, adding a staining solution corresponding to active oxygen to be detected, which is used for immersing a sample, and placing the sample into a vacuum container for low-temperature vacuum staining;
(2) Discarding the dyeing liquid, adding the fixing liquid, and putting the dyeing liquid into a vacuum container for low-temperature vacuum fixing;
(3) Discarding the fixing solution, adding the glycerol aqueous solution, and placing the mixture into a vacuum container for low-temperature vacuum filling and shaping;
(4) Precooling the cold chamber and a slicing cutter to-18 ℃ to-21 ℃, embedding plant root systems of discarded glycerol aqueous solution by using an OTC embedding agent, adjusting the temperature of a fixed table to-18 ℃ to-19 ℃, and slicing;
(5) Sealing the plant root system after the plant root system is warmed.
Preferably, in the steps (1) to (3), the staining solution, the fixing solution and the glycerol aqueous solution are pre-cooled to 0-4 ℃ before use.
As a further preferable aspect, in step (1) to step (3), the staining solution, the fixing solution, and the glycerol aqueous solution are pre-cooled to the same temperature before use.
As a further preferred aspect, in step (1) to step (3), the staining solution, the fixing solution, and the glycerol aqueous solution are pre-cooled to 0 ℃ before use.
Preferably, in the steps (1) to (3), the low-temperature vacuum is specifically: precooling the vacuum container to 0-4deg.C, and vacuumizing to vacuum degree below-0.05 MPa.
Preferably, in the steps (1) to (3), the low-temperature vacuum is specifically: the vacuum vessel was pre-cooled to 0 ℃.
Preferably, in the step (1), the active oxygen to be detected is O 2 - And H 2 O 2 The O is 2 - The dyeing liquid of (a) is as follows: 2.5mmol/L nitrotetrazolium chloride solution in 5mmol/LpH 7.6.6 sodium 3- (n-morpholino) propanesulfonate buffer;
the H is 2 O 2 The dyeing liquid of (a) is as follows: to 100mmol/LpH 6.9.6.9 potassium phosphate buffer, 5units/mL horseradish peroxidase was added, and after mixing, a 2.5mmol/L solution of 3,3' -diaminobenzidine was prepared.
Preferably, in the step (2), the fixing solution is: 5 parts by volume of 38% formaldehyde, 5 parts by volume of glacial acetic acid and 100 parts by volume of 50% alcohol are mixed to prepare the aqueous emulsion.
Preferably, in the step (5), the sealing sheet is formed by using a mixed solution of neutral gum and xylene, wherein the weight ratio of the neutral gum to the xylene in the mixed solution is 1:1.
the beneficial effects of the invention are as follows:
1) The time from material treatment to slice observation is short and can be completed within 12 hours. The original slicing completion time is more than 72 hours, so that a large amount of manufacturing time is saved, and the rapid slicing is realized.
2) The slicing result is accurate, and all the processes from sampling to slicing are completed, so that the low temperature can be maintained, the influence of the temperature on active oxygen metabolism is reduced, and the slicing accuracy is greatly improved.
3) The method adopts the method of vacuum dyeing and freezing the slice, ensures that the tissue cannot shrink, maintains the original life form and ensures the reality and reliability of the slice.
4) Active oxygen is always present in plants, but during stress treatments, physical damage and/or pathogen inoculation, active oxygen bursts intensively. The invention can locate and semi-quantitatively determine the active oxygen in the plant root system, and can presumably evaluate the amount of the active oxygen in the plant root system.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is O after 8h of inoculation of the root system of Lycium barbarum with Fusarium oxysporum 2 - Is used for the histochemical staining of (a).
FIG. 2 shows H after 72H of inoculation of the root system of Lycium barbarum with Fusarium oxysporum 2 O 2 Is used for the histochemical staining of (a).
FIG. 3 shows H after needling the root system of Lycium barbarum and inoculating Fusarium oxysporum for 72H using the prior art method 2 O 2 Is used for the histochemical staining of (a).
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagents.
1. Reagent:
1. detection of O 2 - The dyeing liquid of (a) is as follows: a solution of 2.5mmol/L nitrotetrazolium chloride in sodium 3- (n-morpholino) propanesulfonate buffer of 5mmol/LpH 7.6.6 was prepared.
2. Detection of H 2 O 2 The dyeing liquid of (a) is as follows: to 100mmol/LpH 6.9.6.9 potassium phosphate buffer, 5units/mL horseradish peroxidase was added, and after mixing, a 2.5mmol/L3,3' -diaminobenzidine solution was prepared.
3. Fixing solution: formaldehyde-glacial acetic acid-alcohol (FAA) fixative is prepared by mixing formaldehyde with concentration of 38% of 5mL, glacial acetic acid with concentration of 5mL and alcohol with concentration of 50% of 100 mL.
2. The method for rapidly dyeing and positioning the plant root system active oxygen comprises the following steps:
the method can be used for the rapid reactive oxygen species staining section observation of the plant root system with the diameter of 3-8 mm.
1. Root system sample and treatment
Cutting plant root systems with diameters of 3-8mm into proper lengths, and rapidly fixing the plant root systems to the bottom of a centrifuge tube so as to prevent floating during post-treatment and influence the dyeing fixing effect.
The suitable length may be 0.8-1.2cm.
The quick fixing to the bottom of the centrifuge tube is specifically to quickly fix the sheared plant root system to the bottom of the centrifuge tube by using a weight or a clamp.
2. Root sample dyeing
Adding corresponding active oxygen (active oxygen is O) detected by precooling to 0-4deg.C into the centrifuge tube in step 1 2 - Or H 2 O 2 ) The dyeing liquid of (2) is used for immersing the sample, then the centrifuge tube is vertically placed in a vacuum tank or a vacuum barrel pre-cooled to 0-4 ℃, after the vacuum tank or the vacuum barrel is covered, the vacuum is pumped to reach a negative pressure state, and the dyeing is carried out for 2-4 hours, wherein the specific dyeing time depends on the thickness of the root system sample.
The vacuum pumping can be carried out to a negative pressure state by adjusting the vacuum degree to below-0.05 MPa.
The purpose of low temperature vacuum dyeing is: can ensure that the penetration of the dyeing liquid is enhanced, the dyeing effect is increased and the dyeing time is shortened under the condition of minimal degradation of active oxygen. If only low-temperature dyeing is adopted, the permeability of the dyeing agent is reduced, and the dyeing effect is poor; if only vacuum staining is used, degradation or metabolism of active oxygen components is caused, and the authenticity of the slice is lost.
3. Root system sample fixing
After completion of staining, the centrifuge tube was removed rapidly, and the staining solution was discarded, and buffer solution was used (detection O 2 - Detection of H was performed with 5mmol/L sodium propane sulfonate buffer, pH 7.63- (n-morpholino) 2 O 2 100mmol/LpH, 6.9 potassium phosphate buffer) was washed several times, and fixation was started after washing the residual staining solution.
Adding a fixing solution precooled to 0-4 ℃ into the centrifuge tube, vertically placing the centrifuge tube into a vacuum tank or a vacuum barrel precooled to 0-4 ℃, covering the vacuum tank or the vacuum barrel, vacuumizing to reach a negative pressure state, and fixing for 0.5-1h.
The vacuum pumping can be carried out to a negative pressure state by adjusting the vacuum degree to below-0.05 MPa.
The purpose of the low-temperature vacuum fixation is as follows: can ensure that the fixing effect is enhanced and the fixing time is shortened under the condition of minimum active oxygen degradation. If only a low-temperature fixing agent is adopted, the permeability is reduced, and the fixing effect is poor; if only vacuum fixation is used, degradation or metabolism of active oxygen components is caused, and the authenticity of the slice is lost.
4. Root sample filling shaping
Then discarding the fixing solution, adding glycerol aqueous solution with the volume percentage of 15% pre-cooled to 0-4 ℃, vertically placing the centrifuge tube into a vacuum tank or a vacuum barrel pre-cooled to 0-4 ℃, covering the vacuum tank or the vacuum barrel, vacuumizing to reach a negative pressure state, filling and shaping for 1-3h, filling glycerol in the material tissues, and ensuring the cell morphology.
The vacuum pumping can be carried out to a negative pressure state by adjusting the vacuum degree to below-0.05 MPa.
The purpose of low-temperature vacuum filling and shaping is as follows: can ensure that the shaping effect is enhanced and the shaping time is shortened under the condition of minimal degradation of active oxygen. If only low-temperature filling and shaping are adopted, the permeability of the filling agent is reduced, and the filling speed is low; if only vacuum filling and shaping are adopted, active oxygen components are degraded or metabolized under the influence of high temperature, and the authenticity of the slice is lost.
5. Root system slice
Precooling a cold chamber of a frozen microtome and a slicing tool to-18 ℃ to-21 ℃, vertically placing a root system on a sample support, adding an OTC embedding agent for embedding, finishing the shape to be wedge-shaped after finishing, adjusting the angle to enable the cross section of the sample to be perpendicular to a blade, adjusting the temperature of a fixed table to-18 ℃ to-19 ℃, setting the reasonable thickness of the slice, such as setting the thickness of the slice to 15-25 mu m, and slicing. The plant sample becomes brittle when the temperature is too low, the slices are fragile, and the plant sample is easy to roll when the temperature is too high, so that the plant sample is not suitable for spreading.
6. Slice sealing glue and observation
Dipping the cut sample root section on a glass slide by using a writing brush, taking out the sample root section, standing for 3-5min until the sample returns to the temperature and is completely adhered to the glass slide, sealing the glass slide by using a mixed solution of neutral gum and dimethylbenzene, wherein the weight ratio of the neutral gum to the dimethylbenzene in the mixed solution is 1:1, observed under a microscope and photographed.
The change of temperature and vacuum degree can affect the shrinkage of the plant root tissues, so the precooling temperature and vacuum degree in the steps 2-4 can be kept consistent preferably so as to reduce the shrinkage of the plant root tissues to the greatest extent.
Example 1
The method for rapidly dyeing and positioning the plant root system active oxygen comprises the following steps:
1. root system sample and treatment
Plant roots with the diameter of 5mm are sheared to the length of 1cm, and the roots are quickly fixed to the bottom of a 10mL centrifuge tube by using weights.
2. Root sample dyeing
Pre-cooling a vacuum tank of the vacuum device to 0 ℃, placing a pre-cooling ice bag therein to keep the temperature low, and vertically placing the centrifuge tube in the vacuum tank.
Detection O pre-cooled to 0 ℃ is added 2 - The staining solution of (2) was not passed through the sample.
After the vacuum tank is covered, the vacuum degree is adjusted to minus 0.05MPa, the negative pressure state is reached, and the dyeing is carried out for 2 hours.
3. Root system sample fixing
After the completion of the staining, the centrifuge tube was quickly removed, the staining solution was discarded, and the sample was washed several times with a buffer (buffer: 5mmol/LpH 7.63.63- (n-morpholino) propanesulfonic acid sodium buffer), and the remaining staining solution was washed and then allowed to start to fix.
Adding a fixing solution precooled to 0 ℃ into the centrifuge tube, vertically placing the centrifuge tube into a vacuum tank precooled to 0 ℃, covering the vacuum tank, adjusting the vacuum degree to-0.05 MPa, reaching a negative pressure state, and fixing for 1h.
4. Root sample filling shaping
After discarding the fixing solution, adding 15% glycerol aqueous solution precooled to 0 ℃, vertically placing the centrifuge tube into a vacuum tank precooled to 0 ℃, covering the vacuum tank, adjusting the vacuum degree to-0.05 MPa, reaching a negative pressure state, and filling and shaping for 2h.
5. Root system slice
The cold chamber and the slicing tool were pre-chilled to-21 ℃. Placing a small amount of embedding glue in the center of a cold chamber sample holder, vertically placing a root system on the sample holder, immersing tissues around the sample holder by using a proper amount of bubble-free OTC embedding agent, wherein the total diameter of the embedding agent and the sample is 2 times that of the sample, trimming the embedding glue into a wedge shape after the embedding glue is completely whitened, thinning the embedding glue between a blade and the sample, fixing a cold table on a slicing machine after trimming, adjusting the angle between the sample and the blade, enabling the cross section of the sample to be perpendicular to the blade, setting the thickness of the slice to be 25 mu m, adjusting the temperature of a sample fixing table to be-19 ℃, and slicing.
6. Slice sealing glue and observation
After the root section of the sample is selected, dipping the sample on a glass slide by using a writing brush, taking out the sample, standing the sample for a short time until the sample returns to the temperature and is completely adhered to the glass slide, sealing the glass slide by using a mixed solution of neutral gum and dimethylbenzene, wherein the weight ratio of the neutral gum to the dimethylbenzene in the mixed solution is 1:1, observed under a microscope and photographed.
Taking the medlar root system as an example to carry out the active oxygen O of the plant root system 2 - The specific experiment is as follows:
setting: normal root system group: directly dyeing the root system of the medlar without needling; CK group: dyeing the root system of the medlar after 8 hours of needling; FL group: inoculating fusarium oxysporum after needling, and staining the root system of the medlar after 8 hours; fl+dpi group: after needling, diphenylene iodide chloride (DPI, active oxygen inhibitor) and Fusarium oxysporum are inoculated, and after 8 hours, the root system of the medlar is dyed. Each of the above groups of dyeings uses the method of example 1 of the invention. The experimental results are shown in FIG. 1.
FIG. 1 is O after 8h of inoculation of the root system of Lycium barbarum with Fusarium oxysporum 2 - Is a histochemical staining picture of (c). The upper row is a photograph taken under an optical microscope, O 2 - Dyed blue-violet by nitro tetrazolium chloride (NBT); the lower row is a picture after the dyed color is extracted by Image pro software, and O is arranged in a red line 2 - And positioning the area. Wherein, normal root system group: directly dyeing the root system of the medlar without needling; CK group: dyeing the root system of the medlar after 8 hours of needling; FL group: inoculating fusarium oxysporum after needling, and staining the root system of the medlar after 8 hours; fl+dpi group: after needling, diphenylene iodide chloride (DPI, active oxygen inhibitor) and Fusarium oxysporum are inoculated, and after 8 hours, the root system of the medlar is dyed.
Active oxygen can concentrate and burst during needling or inoculation of pathogenic microorganisms. Active oxygen O 2 - In this example the burst time was 8 hours, different species were treated, and the time of possible bursts was different.
During the dyeing process, the target position to be treated, such as a needling point or an inoculating point, is placed in the middle during shearing, and active oxygen can be generated and dyed because the shearing position is physically damaged.
Example 2
This embodiment differs from embodiment 1 in that: in step 2, the staining solution is used for detecting H 2 O 2 Is a dyeing liquid of (a); in step 3, the buffer solution is: 100mmol/LpH 6.9.9 potassium phosphate buffer. The rest of the procedure is the same as in example 1.
Taking the medlar root system as an example to carry out the active oxygen H of the plant root system 2 O 2 The specific experiment is as follows:
setting: normal root system group: directly dyeing the root system of the medlar without needling; CK group: dyeing the root system of the medlar after needling for 72 hours; FL group: inoculating fusarium oxysporum after needling, and staining the root system of the medlar after 72 hours; fl+dpi group: after needling, diphenylene iodide chloride (DPI, active oxygen inhibitor) and Fusarium oxysporum were inoculated, and after 72 hours, the wolfberry root system was re-stained. Each of the above groups of dyeings uses the method of example 2 of the invention. The experimental results are shown in FIG. 2.
FIG. 2 shows H after 72H of inoculation of the root system of Lycium barbarum with Fusarium oxysporum 2 O 2 Is a histochemical staining picture of (c). The upper row is a photo taken under an optical microscope, H 2 O 2 Dyeing with 3,3' -Diaminobenzidine (DAB) to red; the lower row is a picture after the dyed color is extracted by Image pro software, and H is arranged in a red line 2 O 2 And positioning the area. Wherein, normal root system group: directly dyeing the root system of the medlar without needling; CK group: dyeing the root system of the medlar after needling for 72 hours; FL group: inoculating fusarium oxysporum after needling, and staining the root system of the medlar after 72 hours; fl+dpi group: after needling, diphenylene iodide chloride (DPI, active oxygen inhibitor) and Fusarium oxysporum were inoculated, and after 72 hours, the wolfberry root system was re-stained.
Active oxygen H 2 O 2 In this example the burst time was 72 hours, different species were treated, and the time of possible bursts was different.
During the dyeing process, the target position to be treated, such as a needling point or an inoculating point, is placed in the middle during shearing, and active oxygen can be generated and dyed because the shearing position is physically damaged.
Example 3
The method for rapidly dyeing and positioning the plant root system active oxygen comprises the following steps:
1. root system sample and treatment
Plant roots with the diameter of 8mm are sheared to the length of 0.8cm, and the roots are quickly fixed to the bottom of a 10mL centrifuge tube by using a clamp.
2. Root sample dyeing
Pre-cooling the vacuum barrel to 4 ℃, placing a pre-cooling ice bag therein to keep the temperature low, and vertically placing the centrifuge tube in the vacuum barrel.
Detection O pre-cooled to 4 ℃ is added 2 - The staining solution of (2) was not passed through the sample.
After the vacuum barrel is covered, the vacuum degree is adjusted to minus 0.08MPa, the negative pressure state is reached, and the dyeing is carried out for 4 hours.
3. Root system sample fixing
After the completion of the staining, the centrifuge tube was quickly removed, the staining solution was discarded, and the sample was washed several times with a buffer (buffer: 5mmol/LpH 7.63.63- (n-morpholino) propanesulfonic acid sodium buffer), and the remaining staining solution was washed and then allowed to start to fix.
Adding a fixing solution precooled to 4 ℃ into the centrifuge tube, vertically placing the centrifuge tube into a vacuum barrel precooled to 4 ℃, covering the vacuum barrel, adjusting the vacuum degree to-0.08 MPa, reaching a negative pressure state, and fixing for 1h.
4. Root sample filling shaping
After discarding the fixing solution, adding glycerol water solution with the volume percentage of 15 percent, which is precooled to 4 ℃, vertically placing the centrifuge tube into a vacuum barrel precooled to 4 ℃, covering the vacuum barrel, adjusting the vacuum degree to-0.08 MPa, reaching a negative pressure state, and filling and shaping for 3 hours.
5. Root system slice
The cold chamber and the slicing tool were pre-chilled to-20 ℃. Placing a small amount of embedding glue in the center of a cold chamber sample holder, vertically placing a root system on the sample holder, immersing tissues around the sample holder by using a proper amount of bubble-free OTC embedding agent, wherein the total diameter of the embedding agent and the sample is 2 times that of the sample, trimming the embedding glue into a wedge shape after the embedding glue is completely whitened, thinning the embedding glue between a blade and the sample, fixing a cold table on a slicing machine after trimming, adjusting the angle between the sample and the blade, enabling the cross section of the sample to be perpendicular to the blade, setting the thickness of the slice to be 15 mu m, adjusting the temperature of a sample fixing table to be-19 ℃, and slicing.
6. Slice sealing glue and observation
After the root section of the sample is selected, dipping the sample on a glass slide by using a writing brush, taking out the sample, standing the sample for a short time until the sample returns to the temperature and is completely adhered to the glass slide, sealing the glass slide by using a mixed solution of neutral gum and dimethylbenzene, wherein the weight ratio of the neutral gum to the dimethylbenzene in the mixed solution is 1:1, observed under a microscope and photographed.
Example 4
The method for rapidly dyeing and positioning the plant root system active oxygen comprises the following steps:
1. root system sample and treatment
Plant roots with the diameter of 3mm are sheared to the length of 0.9cm, and the roots are quickly fixed to the bottom of a 10mL centrifuge tube by using a clamp.
2. Root sample dyeing
Pre-cooling the vacuum barrel to 2 ℃, placing a pre-cooling ice bag therein to keep the temperature low, and vertically placing the centrifuge tube in the vacuum barrel.
Detection H by adding precooling to 2 DEG C 2 O 2 The staining solution of (2) was not passed through the sample.
After the vacuum barrel is covered, the vacuum degree is adjusted to minus 0.07MPa, the negative pressure state is reached, and the dyeing is carried out for 3 hours.
3. Root system sample fixing
After the completion of staining, the centrifuge tube was quickly removed, the staining solution was discarded, and the sample was washed several times with a buffer (100 mmol/L of potassium phosphate buffer at pH 6.9), and the remaining staining solution was washed and then allowed to start to fix.
Adding a fixing solution precooled to 2 ℃ into the centrifuge tube, vertically placing the centrifuge tube into a vacuum barrel precooled to 2 ℃, covering the vacuum barrel, adjusting the vacuum degree to-0.07 MPa, reaching a negative pressure state, and fixing for 0.5h.
4. Root sample filling shaping
After discarding the fixing solution, adding glycerol water solution with the volume percentage of 15 percent, which is precooled to 2 ℃, vertically placing the centrifuge tube into a vacuum barrel precooled to 2 ℃, covering the vacuum barrel, adjusting the vacuum degree to-0.07 MPa, reaching a negative pressure state, and filling and shaping for 1h.
5. Root system slice
The cold chamber and the slicing tool were pre-chilled to-18 ℃ prior to slicing. Placing a small amount of embedding glue in the center of a sample holder of a cold chamber, vertically placing a root system on the sample holder, immersing tissues around the sample holder by using a proper amount of bubble-free OTC embedding agent, wherein the total diameter of the embedding agent and the sample is 1.8 times that of the sample, trimming the embedding glue into a wedge shape after the embedding glue is completely whitened, thinning the embedding glue between a blade and the sample, fixing a cold table on a slicing machine after trimming, adjusting the angle between the sample and the blade, enabling the cross section of the sample to be perpendicular to the blade, setting the thickness of the slice to be 20 mu m, adjusting the temperature of a sample fixing table to be-18 ℃, and slicing.
6. Slice sealing glue and observation
After the root section of the sample is selected, dipping the sample on a glass slide by using a writing brush, taking out the sample, standing the sample for a short time until the sample returns to the temperature and is completely adhered to the glass slide, sealing the glass slide by using a mixed solution of neutral gum and dimethylbenzene, wherein the weight ratio of the neutral gum to the dimethylbenzene in the mixed solution is 1:1, observed under a microscope and photographed.
Comparative example 1
The prior art method comprises the following steps: superoxide anion staining: detection of O with the present invention 2 - The dye solution is immersed in the material, and after the material is cultured for 48 hours at normal temperature and normal pressure, the dye solution is discarded, and if the dyeing is slower, the material can be further cultured for 24 hours. Hydrogen peroxide staining: detection of H with the present invention 2 O 2 The dye solution is immersed in the material, and after the material is cultured for 48 hours at normal temperature and normal pressure, the dye solution is discarded, and if the dyeing is slower, the material can be further cultured for 24 hours. After dyeing, the cleaned material is cut into small sections of about 1cm, the small sections are placed into the fixing liquid of the invention to be fixed for 3 to 12 hours at normal temperature and normal pressure, then the small sections are placed into 15 percent glycerol liquid to be soaked for 12 hours at normal temperature and normal pressure, the material is filled between tissues, and then frozen slicing can be carried out. Wherein H is 2 O 2 The staining fixation results are shown in FIG. 3.
FIG. 3 shows H after needling the root system of Lycium barbarum and inoculating Fusarium oxysporum for 72H using the prior art method 2 O 2 Is used for the histochemical staining of (a).
As can be seen from a comparison of fig. 3 and fig. 2: the method has the advantages of long slice staining time, small staining area, serious tissue separation and serious shrinkage.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A rapid dyeing and positioning method for active oxygen of plant root system, wherein the plant is medlar, and is characterized in that: the method comprises the following steps:
(1) Fixing a plant root system to the bottom of a container, adding a staining solution corresponding to active oxygen to be detected, which is used for immersing a sample, and placing the sample into a vacuum container for low-temperature vacuum staining;
(2) Discarding the staining solution, washing the sample for a plurality of times by using the buffer solution, cleaning the residual staining solution, adding the fixing solution, and placing the solution into a vacuum container for low-temperature vacuum fixing;
when detecting O 2 - In the case of the buffer solution, the buffer solution is 5mmol/L of 3- (n-morpholine) propane sodium sulfonate buffer solution with pH of 7.6, and when H is detected 2 O 2 In the process, the buffer solution is 100mmol/L potassium phosphate buffer solution with pH of 6.9;
(3) Discarding the fixing solution, adding the glycerol aqueous solution, and placing the mixture into a vacuum container for low-temperature vacuum filling and shaping;
(4) Precooling the cold chamber and a slicing cutter to-18 ℃ to-21 ℃, embedding plant root systems of discarded glycerol aqueous solution by using an OTC embedding agent, adjusting the temperature of a fixed table to-18 ℃ to-19 ℃, and slicing;
(5) Sealing the plant root system after the plant root system is warmed;
in the step (1) -step (3), the dyeing liquid, the fixing liquid and the glycerol aqueous solution are pre-cooled to 0-4 ℃ before use;
in the step (1) -step (3), the low-temperature vacuum specifically comprises the following steps: precooling the vacuum container to 0-4deg.C, and vacuumizing to vacuum degree below-0.05 MPa.
2. The method according to claim 1, characterized in that: in the step (1) -step (3), the dyeing liquid, the fixing liquid and the glycerol aqueous solution are pre-cooled to the same temperature before use.
3. The method according to claim 2, characterized in that: in the steps (1) to (3), the dyeing liquid, the fixing liquid and the glycerol aqueous solution are pre-cooled to 0 ℃ before use.
4. The method according to claim 1, characterized in that: in the step (1) -step (3), the low-temperature vacuum specifically comprises the following steps: the vacuum vessel was pre-cooled to 0 ℃.
5. The method according to claim 1, characterized in that: in the step (1), the active oxygen to be detected is O 2 - And H 2 O 2 The O is 2 - The dyeing liquid of (a) is as follows: 2.5mmol/L nitrotetrazolium chloride in solution in 5mmol/L sodium 3- (n-morpholino) propanesulfonate buffer at pH 7.6;
the H is 2 O 2 The dyeing liquid of (a) is as follows: to 100mmol/L of potassium phosphate buffer at pH6.9 was added 5units/mL of horseradish peroxidase, and after mixing, a 2.5mmol/L solution of 3,3' -diaminobenzidine was prepared.
6. The method according to claim 1, characterized in that: in the step (2), the fixing liquid is: 5 parts by volume of 38% formaldehyde, 5 parts by volume of glacial acetic acid and 100 parts by volume of 50% alcohol are mixed to prepare the aqueous emulsion.
7. The method according to claim 1, characterized in that: in the step (5), the sealing sheet is formed by using a mixed solution of neutral gum and dimethylbenzene, wherein the weight ratio of the neutral gum to the dimethylbenzene in the mixed solution is 1:1.
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