CN104792806B - A kind of Pollen Tubes metabolic defect in cellular calcium ion ultrastructural Location method - Google Patents
A kind of Pollen Tubes metabolic defect in cellular calcium ion ultrastructural Location method Download PDFInfo
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Abstract
The invention discloses a kind of Pollen Tubes metabolic defect in cellular calcium ion ultrastructural Location method.It is mainly included the following steps that：(1) preparation of qualitative fixer and flushing liquor；(2) material is fixed and rinsed；(3) secondary fixation and flushing；(4) dehydration and transition；(5) resin penetration, embedding, polymerization；(6) material need not dye the micro- sem observation of direct electron.The present invention solves cell turgor regulation, potassium pyroantimonate and buffer components generation abnormal response, material and fixes and post-process and cause pyroantimonic acid calcium precipitate to shift or dilute, dye improper and cover Ca2+The problems such as sediment is observed, completely dispenses with dyeing, also can clear discernable cell device.
Description
Technical field
It is more particularly to a kind of the invention belongs to the film-making of plant cell Ultrastructural observation and chemical composition field of locating technology
Ca in plant pollen solencyte2+Qualitative and position observation technology.
Background technology
Inorganic ions plays important physiological action in plant cell, as adjusted the activity of enzyme, as cyto-architectural
Constituent, regulation intracellular ion balance, Premeabilisation of cells regulation and stomatal movement etc..Ca2+It is not only growth and development of plants
Indispensable element, is also the second messenger of most important regulation cell physiological reaction, and different development signals and environmental stimuli are believed
Number, can all cause Ca including light, mechanical disturbance, abiotic stress, cause of disease exciton etc.2+Change (the Farnklin- of level
Tong, 1999).In the normal growth course of plant and experience environmental stimuli such as light, temperature, biotic, the abiotic side of body
Compel wait reaction in, its numerous cellular physiological processes all with cytoplasm Ca2+Concentration and its characteristic change are relevant.Ca2+With freedom
Ionic state and it is present in plant (Trewavas, 1999), the Ca of multi-form with reference to state2+In the structure and physiology work(of plant
There is its respective effect in energy.Ca2+The deficiency of " pollen population effect " when can compensate in-vitro pollen germination, it is considered to be " flower
The main component of powder growth factor ".Plant cell passes through Ca2+Concentration, amplitude, the change of the frequency that shakes are made to development or outer signals
Reaction.
Ca2+Participate in the vesica secretion process of polytype plant cell, such as pollen tube, plumule protoplast, aleurone
Cell protoplast, Root cap cellll etc..Different types of cell synthesizes to the cell membranes such as polysaccharide, protein, phosphatide and cell membrane
Precursor substance demand it is different, wherein certainly existing extremely complex mechanism to ensure that these materials are accurate in the suitable time
It is really errorless to be transported to correct site (Battey etc., 1996).But up to the present, its mechanism is still unclear
(Blackbourn etc., 1993；Homann and Tester, 1997).Over nearly 20 years, Ca2+The proposition of signal concept with it is widely studied,
Ca in zooblast2+The exploitation of the deep and various advanced experimental technique of signal research, for Ca2+Signal and pollen germination
It is related to that oneself has more in-depth study, but Ca with pollen tube growth2+Effect machine in pollen germination and its pollen tube growth
Reason is still not very clear, and related research is even more rare (Gong et al., 1995).
Potassium pyroantimonate is solvable inorganic compound, into cell after or loose constraint solvable with cell Ca2+Knot
Close, so that pyroantimonic acid calcium precipitate is formed, so as to deposit in the original location.Electron beam can not pass through pyroantimonic acid calcium precipitate, therefore, can
To observe distribution and its form of precipitation under transmission electron microscope.However, a series of asking can be run into practical operation
Topic, such as cell turgor, potassium pyroantimonate and buffer components produce abnormal response, material to fix and post-process and cause pyroantimonic acid
Calcium precipitate is shifted or diluted, dyes improper and cover pyroantimonic acid Ca-deposit observation etc., and influences observation result.
Conventional plant sample Ultrastructural observation film-making is because of the big (~1mm of materials volume3), it is easy to observe；Because of material block institute
It is many containing cell number, choose the target visual field easily, thus conventional plant sample Ultrastructural observation film-making process limiting factor phase
To less.And the pollen tube after pollen and its sprouting belongs to unicellular, pollen pipe diameter is only 20-40 μm, and longitudinal direction is long, how thin
Born of the same parents under slicing conditions while be difficult to find the pollen tubular construction axis position most suitable with activity.
The content of the invention
It is fixed the invention provides a kind of Pollen Tubes metabolic defect in cellular calcium ion ultra microstructure in order to solve above-mentioned technical problem
Position method, the method need not be dyeed, you can observe Ca under transmission electron microscope2+The distribution of precipitation.
The technical solution adopted for solving the technical problem of the present invention is：A kind of Pollen Tubes metabolic defect in cellular calcium ion ultra microstructure
Localization method, it is characterized in that, comprise the following steps：
(1) preparation of solution：
The preparation of 0.2mol/L kaliumphosphate buffers (pH7.8)：Stoste I:Use ddH2O prepares the KH of 0.2mol/L2PO4；It is former
Liquid II：Use ddH2O prepares the K of 0.2mol/L2HPO4·3H2O；By stoste I and stoste II by volume 13:87 mixing；
The preparation of 2.5% glutaraldehyde fixer (pH7.8)：Take the above-mentioned 0.2mol/L kaliumphosphate buffers of new preparation
90ml, adds 2g potassium pyroantimonates, heating for dissolving；After solution is down to room temperature, filtering adds 25% glutaraldehyde solution 10ml, sucrose
1.5g, tannic acid 0.2g, Triton X-100 (triton x-100) 0.5ml, use ddH2O is settled to 100ml, the end of potassium pyroantimonate
Concentration is 2%；
The preparation of dcq buffer liquid：Take 50ml 0.2mol/L kaliumphosphate buffers and add 50ml ddH2O, adds 2g burnt
Potassium antimonate, heating for dissolving after filtering, uses ddH2O is settled to 100ml；
1% osmic acid (OsO4) fixer preparation：The ampoule bottle that 0.5g osmic acids are will be equipped with vent cabinet is cracked, and is put into palm fibre
In color reagent bottle, the above-mentioned dcq buffer liquid 50ml for containing 2% potassium pyroantimonate, as 1% osmic acid fixer is added to be sealed at 4 DEG C
Save backup；
(2) plant pollen tube material 2.5% glutaraldehyde fixer (pH7.8, containing 2% potassium pyroantimonate, 1.5% (w/v) sugarcane
Sugar) 10-24 hours is fixed at 4 DEG C；
(3) after fixed, rinsed 3-5 times, every time 1 hour with the dcq buffer liquid (pH7.8) containing 2% potassium pyroantimonate；
(4) 2-4 hours is fixed again with the 1% osmic acid fixer containing 2% potassium pyroantimonate；
(5) and then with the dcq buffer liquid (pH7.8) containing 2% potassium pyroantimonate rinse 3-5 times, every time 1 hour；
(6) concentration is used to be dehydrated for the series of ethanol of 30%-100% gradients the material after step (5) treatment, ring
Ethylene Oxide transition；
(7) by the material of step (6) through expoxy propane infiltration, Spurr resins+expoxy propane gradient penetration and pure Spurr
Resin penetration 40-48 hours；Then sample (resin+plant pollen tube material) is dripped on slide, polymerization 24 is small at 60 DEG C
When or 65 DEG C at be polymerized 16 hours；
(8) under disecting microscope, the normal pollen tube of growth is chosen, the tree containing target material is cut with sharp blade
Fat block, sticks on another resin-base, repaiies block under anatomical lens again；
(9) cut into slices below side observation in ultramicrotome, choose the section of pollen tube longitudinal center position；Ultra-thin section thickness
It is 60-70nm；
(10) section is not dyeed；Observed under electron microscope, gathers image.
Further, the step (2) is specially：Draw the glutaraldehyde fixers of 3ml 2.5% (pH7.8, containing 2% burnt antimony
Sour potassium, 1.5% (w/v) sucrose) penicillin bottle (bottle closure of rubber) is inserted, plant pollen tube material is inserted in bottle, immediately with note
Material being pumped in emitter insertion bottle stopper and all sinking to bottom, 10-24 hours is fixed at 4 DEG C.
Further, the step (4) is specially：By in material immigration centrifuge tube, 150 μ l 1% are drawn in vent cabinet
In osmic acid (containing 2% potassium pyroantimonate) instillation centrifuge tube, shake up, centrifuge tube is inverted, and fixes 2-4 hours again at room temperature.
Further, the step (6) is specially：By in material immigration penicillin bottle, it is dehydrated step by step with series of ethanol：
30% (30min), 50% (30min), 70% (30min), 85% (20min), 95% (20min), 95% (20min), 100%
(30min), 100% (30min), 100% (60min)；Then transition is permeated with expoxy propane：100% expoxy propane
(20min), 100% expoxy propane (20min).
Further, the step (7) is specially：Material is permeated into 2 hours, Spurr resins+epoxy third with expoxy propane
Alkane (2:1) 3 hours, Spurr resins+expoxy propane (3 are permeated:1) infiltration 3-4 hours, pure Spurr resin penetrations 40-48 hours；
Then sample (resin+pollen tube) is dripped and is polymerized 16 hours on slide, being polymerized 24 hours or 65 DEG C at 60 DEG C；
Remarks：The concentration of above-mentioned ethanol is mass concentration；Ratio between Spurr resins and expoxy propane is volume ratio.
The beneficial effects of the invention are as follows：
(1) glutaraldehyde fixer, osmic acid fixer and flushing liquor all potassium pyroantimonates containing high level, significantly improve
Potassium pyroantimonate and Ca2+Reaction effect, it is to avoid pyroantimonic acid calcium precipitate dilutes；
(2) glutaraldehyde fixes the sucrose containing 1.5% (w/v) in buffer solution, plays a part of to adjust cell turgor, it is to avoid
Cell imbibition and cause eucaryotic cell structure destroy, Ca2+Displacement；
(3) 2%Triton X-100 are added in material fixer, to increase permeability of the cell membrane to fixer molecule,
Significantly improve fixed effect；
(4) traditional ultra-thin section needs uranium acetate to dye 40min, subsequent lead citrate isolation air dyeing 10min.
The present invention cuts into slices without dyeing, it is to avoid lead citrate dyeing is deep, uranium acetate dyeing time is long and sinking of causing
Shallow lake illusion.(final concentration of 0.2%) contains with reference to sucrose, Triton X-100 and height to add tannic acid in the fixed buffer solution of the present invention
The potassium pyroantimonate of amount, completely dispenses with dyeing, also can clear discernable cell device；
(5) 2.5% glutaraldehyde fixers (pH7.8), 2% dcq buffer liquid use preceding filtering, prevent from being precipitated in fixer
Thing contamination of cells, creates a false impression；
(6) ddH is used2O prepares fixed buffer solution and dcq buffer liquid, further reduces pollution.Dcq buffer liquid is flushed
Journey time lengthening, it is to avoid non-specific background pollutes.
Brief description of the drawings
Fig. 1 is Ca in the Wheat Pollen pipe of embodiment 12+Cytochemical localization figure；Wherein, a, b figure respectively pollen tube are thin
The Ca of karyon and vacuole2+Cytochemical localization figure：C figures be control group (2.5% glutaraldehyde fixer without Triton X-100 with
Sucrose) Ca2+Cytochemical localization figure；Scale=0.5 μm；
Fig. 2 is Ca in the blue or green bar pollen tube of embodiment 22+Cytochemical localization figure；Wherein, a figures are the change of pollen tube wall
Learn positioning figure：B figures are the Ca of control group (2.5% glutaraldehyde fixer is without Triton X-100 and sucrose)2+Cytochemistry is determined
Bitmap；Scale=0.5 μm.
Specific embodiment
Embodiment 1
(1) preparation of solution：
The preparation of 0.2mol/L kaliumphosphate buffers (pH7.8)：Stoste I:By 2.72g KH2PO4It is dissolved in 100ml ddH2O；
Stoste II：By 4.56g K2HPO4·3H2O is dissolved in 100ml ddH2In O, then by stoste I and II by volume 13:87 mixing；
2.5% glutaraldehyde fixer (pH7.8)：The above-mentioned 0.2mol/L kaliumphosphate buffers 90ml of new preparation is taken, is added
2g potassium pyroantimonates, heating for dissolving；After solution is down to room temperature, filtering adds 25% glutaraldehyde solution 10ml, sucrose 1.5g, tannic acid
0.2g, Triton X-1000.5ml, use ddH2O is settled to 100ml, final concentration of the 2% of potassium pyroantimonate；
The preparation of dcq buffer liquid：Take 50ml 0.2mol/L kaliumphosphate buffers (pH7.8) and add 50ml ddH2O, then
2g potassium pyroantimonates are added, heating for dissolving after filtering, uses ddH2O is settled to 100ml；
1% osmic acid (OsO4) fixer preparation：The ampoule bottle that 0.5g osmic acids are will be equipped with vent cabinet is cracked, and is put into palm fibre
In color reagent bottle, the above-mentioned dcq buffer liquid 50ml for containing 2% potassium pyroantimonate, as 1% osmic acid fixer is added to be sealed at 4 DEG C
Save backup；
(2) the glutaraldehyde fixers of 3ml 2.5% (pH7.8, containing 2% potassium pyroantimonate, 1.5% (w/v) sucrose) is drawn to insert
The penicillin bottle (bottle closure of rubber) of 5ml or 10ml, Wheat Pollen pipe is inserted in bottle, is inserted in bottle stopper with syringe be evacuated immediately
Bottom is all sunk to material, 10 hours are fixed at 4 DEG C；
(3) after fixed, rinsed 4 times, every time 1 hour with the dcq buffer liquid (pH7.8) containing 2% potassium pyroantimonate；
(4) the osmic acid fixers of 150 μ l 1% in material immigration 1ml centrifuge tubes, will be drawn in vent cabinet (containing 2% burnt antimony
Sour potassium) instill centrifuge tube in, shake up, centrifuge tube be inverted, fix 4 hours again at room temperature；
(5) sample is rinsed 4 times, every time 1 hour with the dcq buffer liquid (pH7.8) containing 2% potassium pyroantimonate；
(6) by material move into penicillin bottle in, with 30% (30min), 50% (30min), 70% (30min), 85%
(20min), 95% (20min), 95% (20min), 100% (30min), 100% (30min), 100% (60min) series second
Alcohol is dehydrated step by step, 100% expoxy propane (20min), 100% expoxy propane (20min) infiltration transition；
(7) material is permeated into 2 hours, Spurr resins+expoxy propane (2 with expoxy propane:1) 3 hours, Spurr trees are permeated
Fat+expoxy propane (3:1) infiltration 3 hours, pure Spurr resin penetrations 40 hours；Then sample (resin+pollen tube) is dripped in load
On slide, it is polymerized 24 hours at 60 DEG C；
(8) under disecting microscope, the normal pollen tube of growth is chosen, is cut containing target pollen tube with sharp blade
Resin, sticks on another resin-base after repairing block, repaiies block under anatomical lens again；
(9) cut into slices below side observation in ultramicrotome, choose the section of pollen tube longitudinal center position；Ultra-thin section thickness
It is 70nm；
(10) section is not dyeed, and observed under electron microscope gathers image；
(11) above-mentioned operation repetitive is pressed with 2.5% glutaraldehyde fixer of sucrose with without Triton X-100, as right
According to.
Result is as shown in figure 1, pyroantimonic acid calcium precipitate is distributed mainly in pollen tube nucleus (a) and vacuole (b).Control group
The Ca of (2.5% glutaraldehyde fixer is without Triton X-100 and sucrose)2+Cytochemical localization shows, thin due to that can not adjust
Born of the same parents' turgescence, Ca2+Extravasation, causes to produce Ca outside pollen tube wall2+Precipitation, and pollen tube internal precipitate is rare, precipitate colour developing shallow (c).
Embodiment 2
(1) preparation of solution：
The preparation of 0.2mol/L kaliumphosphate buffers (pH7.8)：Stoste I:By 2.72g KH2PO4It is dissolved in 100ml ddH2O；
Stoste II：By 4.56g K2HPO4·3H2O is dissolved in 100ml ddH2In O, then by stoste I and II by volume 13:87 mixing；
2.5% glutaraldehyde fixer (pH7.8)：The above-mentioned 0.2mol/L kaliumphosphate buffers 90ml of new preparation is taken, is added
2g potassium pyroantimonates, heating for dissolving；After solution is down to room temperature, filtering adds 25% glutaraldehyde solution 10ml, sucrose 1.5g, tannic acid
0.2g, Triton X-1000.5ml, use ddH2O is settled to 100ml, final concentration of the 2% of potassium pyroantimonate；
The preparation of dcq buffer liquid：50ml 0.2mol/L kaliumphosphate buffers (pH7.8) add 50ml ddH2O, then add
Enter 2g potassium pyroantimonates, heating for dissolving after filtering, uses ddH2O is settled to 100ml；
1% osmic acid (OsO4) fixer preparation：The ampoule bottle that 0.5g osmic acids are will be equipped with vent cabinet is cracked, and is put into palm fibre
In color reagent bottle, the above-mentioned dcq buffer liquid 50ml for containing 2% potassium pyroantimonate, as 1% osmic acid fixer is added to be sealed at 4 DEG C
Save backup；
(2) the glutaraldehyde fixers of 3ml 2.5% (pH7.8, containing 2% potassium pyroantimonate, 1.5% (w/v) sucrose) insert 5ml or
The penicillin bottle (bottle closure of rubber) of 10ml, Qing Wilsonii pollen tubes are inserted in bottle, inserted in bottle stopper with syringe be pumped to material immediately
Material all sinks to bottom, and 10 hours are fixed at 4 DEG C；
(3) after fixed, rinsed 5 times, every time 1 hour with the dcq buffer liquid (pH7.8) containing 2% potassium pyroantimonate；
(3) the osmic acid fixers of 150 μ l 1% in material immigration 1ml centrifuge tubes, will be drawn in vent cabinet (containing 2% burnt antimony
Sour potassium) instill centrifuge tube in, shake up, centrifuge tube be inverted, fix 4 hours again at room temperature；
(4) sample is rinsed 5 times, every time 1 hour with the dcq buffer liquid (pH7.8) containing 2% potassium pyroantimonate；
(5) by material move into penicillin bottle in, with 30% (30min), 50% (30min), 70% (30min), 85%
(20min), 95% (20min), 95% (20min), 100% (30min), 100% (30min), 100% (60min) series second
Alcohol is dehydrated step by step, 100% expoxy propane (20min), 100% expoxy propane (20min) infiltration transition；
(6) material is permeated into 2 hours, Spurr resins+expoxy propane (2 with expoxy propane:1) 3 hours, Spurr trees are permeated
Fat+expoxy propane (3:1) infiltration 4 hours, pure Spurr resin penetrations 48 hours；Then sample (resin+pollen tube) is dripped in load
On slide, it is polymerized 16 hours at 65 DEG C；
(7) under disecting microscope, the normal pollen tube of growth is chosen, is cut containing target pollen tube with sharp blade
Resin, sticks on another resin-base after repairing block, repaiies block under anatomical lens again；
(8) cut into slices below side observation in ultramicrotome, choose the section of pollen tube longitudinal center position；Ultra-thin section thickness
It is 60nm；
(9) section is not dyeed, and observed under electron microscope gathers image；
(10) above-mentioned operation repetitive is pressed with 2.5% glutaraldehyde fixer of sucrose with without Triton X-100, as right
According to.
Result is as shown in Figure 2.Pyroantimonic acid calcium precipitate is distributed mainly on pollen tube wall (a).Control group (consolidate by 2.5% glutaraldehyde
Determine liquid without Triton X-100 and sucrose) Ca2+Cytochemical localization shows, due to pollen tube cell turgor can not be adjusted,
Ca2+Extravasation, causes to produce Ca outside pollen tube wall2+Precipitation, and rare, precipitation is precipitated in pollen tube wall and develops the color shallow (b).
Claims (5)
1. a kind of Pollen Tubes metabolic defect in cellular calcium ion ultrastructural Location method, it is characterized in that, comprise the following steps：
(1) preparation of solution：
The preparation of 0.2mol/L kaliumphosphate buffers：Stoste I:Use ddH2O prepares the KH of 0.2mol/L2PO4；Stoste II：Use ddH2O
Prepare the K of 0.2mol/L2HPO4·3H2O；By stoste I and stoste II by volume 13:87 mixing；
The preparation of 2.5% glutaraldehyde fixer：The above-mentioned 0.2mol/L kaliumphosphate buffers 90ml of new preparation is taken, 2g Jiao's antimony is added
Sour potassium, heating for dissolving；After solution is down to room temperature, filtering, add 25% glutaraldehyde solution 10ml, sucrose 1.5g, tannic acid 0.2g,
Triton X-100 0.5ml, use ddH2O is settled to 100ml；
The preparation of dcq buffer liquid：Take 50ml 0.2mol/L kaliumphosphate buffers and add 50ml ddH2O, adds 2g pyroantimonic acids
Potassium, heating for dissolving after filtering, uses ddH2O is settled to 100ml；
The preparation of 1% osmic acid fixer：Take 0.5g osmic acids and add above-mentioned dcq buffer liquid 50ml, as 1% osmic acid fixer；
(2) plant pollen tube material fixes 10-24 hours with 2.5% glutaraldehyde fixer at 4 DEG C；
(3) after fixed, rinsed 3-5 times with dcq buffer liquid；
(4) 2-4 hours is fixed again with 1% osmic acid fixer；Specially：By in material immigration 1ml centrifuge tubes, 150 μ l are drawn
In 1% osmic acid fixer instillation centrifuge tube, shake up, centrifuge tube is inverted, and fixes 2-4 hours again at room temperature；
(5) and then with dcq buffer liquid rinse 3-5 times；
(6) concentration of alcohol is used to be dehydrated for the series of ethanol of 30%-100% gradients the material after step (5) treatment, ring
Ethylene Oxide transition；
(7) by the material of step (6) through expoxy propane infiltration, Spurr resins+expoxy propane gradient penetration and pure Spurr resins
Infiltration 40-48 hours；Then resin+Pollen Tubes material sample is dripped on slide, be polymerized at 60 DEG C 24 hours or 65
It is polymerized 16 hours at DEG C；
(8) under disecting microscope, the normal pollen tube of growth is chosen, the resin containing target material is cut with sharp blade
Block, sticks on another resin-base, repaiies block under anatomical lens again；
(9) cut into slices below side observation in ultramicrotome, choose the section of pollen tube longitudinal center position；Ultra-thin section thickness is
60-70nm；
(10) section is not dyeed；Observed under electron microscope, gathers image.
2. a kind of Pollen Tubes metabolic defect in cellular calcium ion ultrastructural Location method as claimed in claim 1, it is characterized in that, it is described
Step (2) is specially：Draw the glutaraldehyde fixers of 3ml 2.5% and insert penicillin bottle, plant pollen tube material is inserted in bottle,
Inserted with syringe immediately and be pumped to material and all sink to bottom in bottle stopper, 10-24 hours is fixed at 4 DEG C.
3. a kind of Pollen Tubes metabolic defect in cellular calcium ion ultrastructural Location method as claimed in claim 1, it is characterized in that, it is described
Step (6) is specially：By in material immigration penicillin bottle, it is dehydrated step by step with series of ethanol：30% Ethanol Treatment 30min,
50% Ethanol Treatment 30min, 70% Ethanol Treatment 30min, 85% Ethanol Treatment 20min, 95% Ethanol Treatment
20min, 95% Ethanol Treatment 20min, 100% Ethanol Treatment 30min, 100% Ethanol Treatment 30min, 100% second
Alcohol processes 60min；Then transition is permeated with expoxy propane：100% expoxy propane treatment 20min, the treatment of 100% expoxy propane
20min。
4. a kind of Pollen Tubes metabolic defect in cellular calcium ion ultrastructural Location method as claimed in claim 1, it is characterized in that, it is described
Step (7) is specially：It is 2 by material expoxy propane infiltration 2 hours, volume ratio：1 Spurr resins+expoxy propane infiltration 3
Hour, volume ratio are 3:1 Spurr resins+expoxy propane infiltration 3-4 hours, pure Spurr resin penetrations 40-48 hours；Then
Resin+Pollen Tubes material sample is dripped and is polymerized 16 hours on slide, being polymerized 24 hours or 65 DEG C at 60 DEG C.
5. a kind of Pollen Tubes metabolic defect in cellular calcium ion ultrastructural Location method as described in any one in claim 1-4,
It is characterized in that, the step (3), each washing time of (5) they are 1 hour.
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| CN107505175B (en) * | 2017-07-12 | 2020-02-04 | 青岛农业大学 | Method for ultrathin slicing of tip of pollen tube |
| CN108827741A (en) * | 2018-07-19 | 2018-11-16 | 青岛农业大学 | A method of fluorescent dye is loaded in pollen tube using negative-pressure penetration |
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| CN111157433A (en) * | 2020-01-06 | 2020-05-15 | 中国科学院生态环境研究中心 | Kit for marking single cell of red blood cell and detection method thereof |
| CN112393964B (en) * | 2020-11-16 | 2021-07-27 | 南京农业大学 | Preparation method of biological single cell transmission electron microscope sample |
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