CN102692497A - Method for observing cell microtubule framework through soybean root tip treatment by immunofluorescence staining - Google Patents
Method for observing cell microtubule framework through soybean root tip treatment by immunofluorescence staining Download PDFInfo
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- CN102692497A CN102692497A CN2012101914449A CN201210191444A CN102692497A CN 102692497 A CN102692497 A CN 102692497A CN 2012101914449 A CN2012101914449 A CN 2012101914449A CN 201210191444 A CN201210191444 A CN 201210191444A CN 102692497 A CN102692497 A CN 102692497A
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Abstract
The invention discloses a method for observing a cell microtubule framework through soybean root tip treatment by immunofluorescence staining. An immunofluorescence staining method is used for treating soybean root tips grown for 1 to 10 days, and a confocal microscopy is used for observing the cell microtubule framework of the root tip cortex. The method is simple, convenient, accurate and fast.
Description
Technical field
The present invention relates to a kind of method of handling soybean tip of a root observation of cell microtubule skeleton with immunofluorescence dyeing; Be specifically related to a kind of immunofluorescence staining that utilizes and handle the method that the soybean tip of a root is observed the root-tip cells microtubule skeleton, belong to plant protection and cell biology field.
Background technology
Pathogen and its host do to be considered to an arms race mutually, and in this contest, pathogen is through various policies using or suppress host's defense reaction, and host itself also utilizes various strategy identifications and attacks the pathogen or the pathogen effector molecule of invasion.Pathogen and non-host do mutually and and the definition difference of host between doing mutually be that pathogen overcomes the ability that a series of obstacles are successfully invaded the host.PLANT CYTOSKELETON often is considered to pathogen and invades one of one obstacle of host.The importance of cytoskeleton in resisting the pathogen phagocytic process can be passed through some example shows, can be penetrated by many non-host fungies after being destroyed like the actin cytoskeleton of barley, wheat, cucumber and tobacco.Pathogen is successfully invaded the host usually need be through 6 road obstacles.More and more evidences shows, cytoskeleton is participated in is the 3rd road obstacle that the host forms when invaded by pathogen, mainly is to induce such as mastoid process and callose formation, albumen and carbohydrates to accumulate even the generation of obstacles such as anaphylaxis cell death.The indirect person that PLANT CYTOSKELETON is formed, transported as intracellular organic matter by actin fibril and microtubule is playing the part of crucial effects.Nineteen nineties has been reported the research of cytoskeleton effect in the first example description plant defense reaction in early days, finds when plant receives the pathogen intrusion a large amount of host cell polar.In the vegetable cell that receives the pathogen intrusion, it is consistent with the nucleus moving direction to observe the tenuigenin convergence point, promptly all moves to p of E.People such as Kobayashi find when utilizing the method for immunocytochemistry to study the barley germs sheath cell microtubule institutional framework of non-host's cause of disease pea powdery mildew (Erysiphe pisi) when infecting barley; In the host cell that does not receive pathogen infection; Microtubule becomes transversely arranged along the longitudinal axis of cell; And in by the cell of pathogen infection, microtubule p of E be gathered into the radiation netted.Utilize same immunofluorescence technique also observes the host in a series of host who comprises the rotten grape spore of flax and flax rest fungus, soybean and soybean blight bacterium, onion and green onion and barley and big wheat powdery mildew and the affine mutual work of pathogen microtubule and flesh kinetodesma towards fungi with the oomycetes p of E redistributes and arrangement.The barley germs sheath cell is compared with the non-pathogenic powdery mildew of barley mutually; The speed that microtubule and actin fibril are arranged again in the affine mutual work of barley germs sheath cell and pathogenic powdery mildew is obviously slowed down; Equally similar results has also been found in other pathogenic systematic research; Clear evidence has been supported the effect of PLANT CYTOSKELETON in the plant defense reaction, also shows the arrangement again of affine pathogen meeting interference cell skeleton simultaneously.
Summary of the invention
The objective of the invention is to overcome the prior art deficiency, and provide a kind of quick, easy, observe the method for soybean root-tip cells microtubule skeleton with the immunofluorescence dyeing method exactly.
The technical scheme of invention is:
Keep existing immunofluorescence dyeing and observational technique constant; The exciting light that comprises fluorescence antibody kind and consumption, used Laser Scanning Confocal Microscope is all identical with routine with the wavelength of transmitted light scope; Handle 1-10 days the soybean tip of a root with the immunofluorescence dyeing method, Laser Scanning Confocal Microscope is observed tip of a root cortical cell microtubule skeleton; Said immunofluorescence dyeing method disposal route is: the soybean tip of a root is put into the immobile liquid 10min that bleeds, and room temperature is 1hr fixedly; It is inferior to give a baby a bath on the third day after its birth with PEMT after fixedly finishing, each 10min; Enzymolysis 1hr under 37 ℃ of conditions; The PEM flushing is three times behind the enzymolysis, each 10min; With 0.1M PBS (pH7.2) solution preparation NaBH4, making its concentration is 1mg/ml, and the NaBH4 with the 1mg/ml for preparing handles soybean tip of a root 20min again; Room temperature is with 50mM Gly sealing 30min; Add one and resist, 4 ℃ are spent the night, and flushing one is anti-: PBS flushing three times, each 10min; Add two anti-(1: 600), under 37 ℃, black out condition, 3hr; Flushing two is anti-: PBS flushing three times, each 10min; After adding anti-fluorescent quenching agent, mounting.
Beneficial effect of the present invention is:
Method is easy, accurate, quick.Utilize the method for immunofluorescence dyeing to handle the soybean tip of a root, Laser Scanning Confocal Microscope is observed tip of a root cortical cell microtubule skeleton, can directly grasp soybean root-tip cells microtubule skeleton arrangement mode qualitatively.Immunofluorescence staining is directly handled and is observed paddy rice root tip cell microtubule skeleton and arranges and can effectively avoid handling and complex steps and receive residual chlorophyllous interference easily when observing the blade cell microtubule skeleton such as immunofluorescence dyeing, organizes cell microtubule skeleton when doing mutually for further research pathogenic soil bacterium and plant roots simultaneously and to the observation of pathogen response effectively simply method is provided.
Embodiment
Below in conjunction with specific embodiment, the present invention is elaborated.
Embodiment one:
Immunofluorescence staining is handled 1 day the soybean tip of a root, and Laser Scanning Confocal Microscope is observed tip of a root cortical cell microtubule skeleton.
Concrete grammar is: the soybean tip of a root is put into immobile liquid (number percent is mass percent for 4% paraformaldehyde, 0.1% glutaraldehyde, the down together) 10min that bleeds, and room temperature is 1hr fixedly.It is inferior to give a baby a bath on the third day after its birth with PEMT (0.05%Triton x-100) after fixedly finishing, each 10min.Enzymolysis 1hr under 37 ℃ of conditions, PEM preparation, 1% pectase, 1.5% cellulase, 0.4M sweet mellow wine.The PEM flushing is three times behind the enzymolysis, each 10min.With 0.1M PBS (pH7.2) solution preparation NaBH4, making its concentration is 1mg/ml, and (damping fluid of PBS preparation NaBH4) NaBH4 with the 1mg/ml for preparing again handled soybean tip of a root 20min.Room temperature is with 50mM Gly sealing 30min.Added for one anti-(β-tubulin one resists 1: 800, and AtMAP18 one resists 1: 100), 4 ℃ are spent the night, and flushing one is anti-: PBS flushing three times, 10min at every turn.Add two anti-(1: 600), under 37 ℃, black out condition, 3hr.Flushing two is anti-: PBS flushing three times, each 10min.After adding anti-fluorescent quenching agent, mounting.The result has observed microtubule skeleton at the cortical cell of root cap, meristematic zone and the elongation zone of the tip of a root.
Embodiment two:
Immunofluorescence staining is handled 3 days the soybean tip of a root, and Laser Scanning Confocal Microscope is observed tip of a root cortical cell microtubule skeleton.
The soybean tip of a root is put into immobile liquid (4% paraformaldehyde, the 0.1% glutaraldehyde) 10min that bleeds, and room temperature is 1hr fixedly.It is inferior to give a baby a bath on the third day after its birth with PEMT (0.05%Triton x-100) after fixedly finishing, each 10min.Enzymolysis 1hr under 37 ℃ of conditions, PEM preparation: 1% pectase, 1.5% cellulase, 0.4M sweet mellow wine.The PEM flushing is three times behind the enzymolysis, each 10min.Handle 20min with 1mg/ml NaBH4 (PBS preparation).Room temperature is with 50mM Gly sealing 30min.Added for one anti-(β-tubulin one resists 1: 800, and AtMAP18 one resists 1: 100), 4 ℃ of flushings one of spending the night are anti-: PBS flushing three times, 10min at every turn.Add two anti-(1: 600), under 37 ℃, black out condition, 3hr.Flushing two is anti-: PBS flushing three times, each 10min.After adding anti-fluorescent quenching agent, mounting.The result has observed microtubule skeleton at the cortical cell of root cap, meristematic zone and the elongation zone of the tip of a root.
Embodiment three:
Immunofluorescence staining is handled 5 days the soybean tip of a root, and Laser Scanning Confocal Microscope is observed tip of a root cortical cell microtubule skeleton.
The soybean tip of a root is put into immobile liquid (4% paraformaldehyde, the 0.1% glutaraldehyde) 10min that bleeds, and room temperature is 1hr fixedly.It is inferior to give a baby a bath on the third day after its birth with PEMT (0.05%Triton x-100) after fixedly finishing, each 10min.Enzymolysis 1hr under 37 ℃ of conditions, PEM preparation, 1% pectase, 1.5% cellulase, 0.4M sweet mellow wine.The PEM flushing is three times behind the enzymolysis, each 10min.Handle 20min with 1mg/ml NaBH4 (PBS preparation).Room temperature is with 50mM Gly sealing 30min.Added for one anti-(β-tubulin one resists 1: 800, and AtMAP18 one resists 1: 100), 4 ℃ of flushings one of spending the night are anti-: PBS flushing three times, 10min at every turn.Add two anti-(1: 600), under 37 ℃, black out condition, 3hr.Flushing two is anti-: PBS flushing three times, each 10min.After adding anti-fluorescent quenching agent, mounting.The result has observed microtubule skeleton at the cortical cell of root cap, meristematic zone and the elongation zone of the tip of a root.
Embodiment four:
Immunofluorescence staining is handled 7 days the soybean tip of a root, and Laser Scanning Confocal Microscope is observed tip of a root cortical cell microtubule skeleton.
The soybean tip of a root is put into immobile liquid (4% paraformaldehyde, the 0.1% glutaraldehyde) 10min that bleeds, and room temperature is 1hr fixedly.It is inferior to give a baby a bath on the third day after its birth with PEMT (0.05%Triton x-100) after fixedly finishing, each 10min.Enzymolysis 1hr PEM preparation under 37 ℃ of conditions, 1% pectase, 1.5% cellulase, 0.4M sweet mellow wine.The PEM flushing is three times behind the enzymolysis, each 10min.Handle 20min with 1mg/ml NaBH4 (PBS preparation).Room temperature is with 50mM Gly sealing 30min.Added for one anti-(β-tubulin one resists 1: 800, and AtMAP18 one resists 1: 100), 4 ℃ of flushings one of spending the night are anti-: PBS flushing three times, 10min at every turn.Add two anti-(1: 600), under 37 ℃, black out condition, 3hr.Flushing two is anti-: PBS flushing three times, each 10min.After adding anti-fluorescent quenching agent, mounting.The result has observed microtubule skeleton at the cortical cell of root cap, meristematic zone and the elongation zone of the tip of a root.
Embodiment five:
Immunofluorescence staining is handled 10 days the soybean tip of a root, and Laser Scanning Confocal Microscope is observed tip of a root cortical cell microtubule skeleton.
The soybean tip of a root is put into immobile liquid (4% paraformaldehyde, the 0.1% glutaraldehyde) 10min that bleeds, and room temperature is 1hr fixedly.It is inferior to give a baby a bath on the third day after its birth with PEMT (0.05%Triton x-100) after fixedly finishing, each 10min.Enzymolysis 1hr PEM preparation under 37 ℃ of conditions, 1% pectase, 1.5% cellulase, 0.4M sweet mellow wine.The PEM flushing is three times behind the enzymolysis, each 10min.Handle 20min with 1mg/ml NaBH4 (PBS preparation).Room temperature is with 50mM Gly sealing 30min.Added for one anti-(β-tubulin one resists 1: 800, and AtMAP18 one resists 1: 100), 4 ℃ of flushings one of spending the night are anti-: PBS flushing three times, 10min at every turn.Add two anti-(1: 600), under 37 ℃, black out condition, 3hr.Flushing two is anti-: PBS flushing three times, each 10min.After adding anti-fluorescent quenching agent, mounting.The result has observed microtubule skeleton at the cortical cell of root cap, meristematic zone and the elongation zone of the tip of a root.
Should be understood that, concerning those of ordinary skills, can improve or conversion, and all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention according to above-mentioned explanation.
Claims (1)
1. the method with immunofluorescence dyeing processing soybean tip of a root observation of cell microtubule skeleton is characterized in that handle 1-10 days the soybean tip of a root with the immunofluorescence dyeing method, Laser Scanning Confocal Microscope is observed tip of a root cortical cell microtubule skeleton; Said immunofluorescence dyeing method disposal route is: the soybean tip of a root is put into the immobile liquid 10min that bleeds, and room temperature is 1hr fixedly; It is inferior to give a baby a bath on the third day after its birth with PEMT after fixedly finishing, each 10min; Enzymolysis 1hr under 37 ℃ of conditions; The PEM flushing is three times behind the enzymolysis, each 10min; With 0.1M PBS (pH7.2) solution preparation NaBH4, making its concentration is 1mg/ml, and the NaBH4 with the 1mg/ml for preparing handles soybean tip of a root 20min again; Room temperature is with 50mM Gly sealing 30min; Add one and resist, 4 ℃ are spent the night, and flushing one is anti-: PBS flushing three times, each 10min; Add two anti-(1: 600), under 37 ℃, black out condition, 3hr; Flushing two is anti-: PBS flushing three times, each 10min; After adding anti-fluorescent quenching agent, mounting.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103267727A (en) * | 2013-05-20 | 2013-08-28 | 云南农业大学 | Method for detecting piricularia oryzae effect protein transferring in rice root tissue |
| CN103439500A (en) * | 2013-08-27 | 2013-12-11 | 陕西科技大学 | Visible tracking and detecting method for protease in leather treatment process |
| CN103528872A (en) * | 2013-10-14 | 2014-01-22 | 中国农业大学 | Fluorescent staining technique of plant root tissue living body section and application of technique |
| CN107315008A (en) * | 2017-03-15 | 2017-11-03 | 信阳师范学院 | A kind of method for observing paddy rice root tip micro-pipe band arrangement mode |
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| CN1840704A (en) * | 2006-01-26 | 2006-10-04 | 中国农业科学院棉花研究所 | Cotton in-situ hybridization method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267727A (en) * | 2013-05-20 | 2013-08-28 | 云南农业大学 | Method for detecting piricularia oryzae effect protein transferring in rice root tissue |
| CN103267727B (en) * | 2013-05-20 | 2016-03-16 | 云南农业大学 | A kind of method detecting Pyricularia oryzae effect protein and transport in rice root tissue |
| CN103439500A (en) * | 2013-08-27 | 2013-12-11 | 陕西科技大学 | Visible tracking and detecting method for protease in leather treatment process |
| CN103439500B (en) * | 2013-08-27 | 2015-01-28 | 陕西科技大学 | Visible tracking and detecting method for protease in leather treatment process |
| CN103528872A (en) * | 2013-10-14 | 2014-01-22 | 中国农业大学 | Fluorescent staining technique of plant root tissue living body section and application of technique |
| CN107315008A (en) * | 2017-03-15 | 2017-11-03 | 信阳师范学院 | A kind of method for observing paddy rice root tip micro-pipe band arrangement mode |
| CN107315008B (en) * | 2017-03-15 | 2019-12-03 | 信阳师范学院 | A method of observation paddy rice root tip micro-pipe band arrangement mode |
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Application publication date: 20120926 |