CN106771129A - The Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton - Google Patents

Abstract

The invention provides a kind of Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton, comprise the following steps that：The steps such as fixer is fixed, is dehydrated, the removal of freezing microtome section, non-specific fluorescence, immune labeled and mounting, the present invention is combined with the microtubule skeleton at the tender position of Young Cotton by fluorescein, cotton tender tissue microtubule skeleton is carried out immune labeled, the change of tender tissue microtubule skeleton is observed from tissue level, cotton is studied the resistance of pest and disease damage the resistance of abiotic stress and cotton.Step of the present invention is simple, it is easy to grasp, and as a result clearly, is adapted to observe cotton tender tissue microtubule skeleton.Also there is certain reference value to the observation of other plant tender tissue microtubule skeleton.

Description

Immunofluorescent Antibody Labeling of Microtubule Skeleton in Young Cotton Tissue

Technical field:

The invention belongs to the technical field of cotton scientific research, and in particular relates to an immunofluorescent antibody labeling method for the microtubule skeleton of young cotton tissues.

Background technique:

Cotton is one of the important economic crops in my country. Some abiotic stresses such as low temperature, drought and salt damage seriously affect the growth and development of cotton. At the same time, pests and diseases are also one of the important reasons affecting the growth and development of cotton. Stress resistance and cotton's resistance to diseases and insect pests are important contents of scientific research on cotton by researchers.

Fluorescent antibody labeling technology is to chemically combine fluorescein with specific antibodies to form a fluorescein-protein conjugate, that is, a fluorescently labeled antibody. This conjugate still retains the activity of the antibody and has the disappearance of fluorescein. After it is specifically combined with the corresponding antigen, it will show bright specific fluorescence by means of fluorescence microscope observation. When studying the resistance of cotton to abiotic stress and the resistance of cotton to diseases and insect pests, fluorescent antibody labeling technology is usually used.

At present, when using fluorescent antibody labeling technology to study cotton, the fluorescent antibody labeling technology is mainly used to label cotton pollen grains, protoplasts, and individual plant root tip cells dissociated by enzymatic hydrolysis. However, fluorescent antibodies have not yet been labeled on the tissue level of cotton, such as the microtubule skeleton at the shoot tip and the microtubule skeleton at the root tip of cotton. Microtubules are an important component of the cytoskeleton. The microtubule skeleton and the associated microtubule-binding proteins are widely involved in the maintenance of cell shape, cell elongation and division, material transport, cell differentiation and other life activities. At the same time, it can also be used as a signal substrate to participate in plant growth and development, plant disease interaction, plant and adversity stress, etc.

Invention content:

In summary, in order to overcome the deficiencies of the existing technical problems, the present invention provides a method for immunofluorescent antibody labeling of the microtubule skeleton of young cotton tissues, which is to combine fluorescein with the microtubule skeleton of cotton tender parts Combined with immunolabeling of the microtubule skeleton of young cotton tissues, observing the changes of the microtubule skeleton of young tissues at the tissue level, and studying the resistance of cotton to abiotic stress and the resistance of cotton to diseases and insect pests.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

A method for immunofluorescent antibody labeling of cotton young tissue microtubule skeleton, wherein: comprising the following process steps:

The first step, fixative fixation

Cut the young tissue of cotton, put the young tissue into a sealed container with fixative, vacuumize the sealed container for 8-15 minutes, then let the sealed container stand at 25-30°C for 1.5-3 hours, The fixative in the sealed container fixes the tender tissue of cotton;

The second step, dehydration

Taking out the tender tissue of cotton in the airtight container, and then putting the tender tissue into sucrose phosphate buffer solution for soaking and dehydration;

Step 3: Frozen Sectioning

Put the dehydrated cotton tender tissue into a cryostat for sectioning, the internal temperature of the cryostat is -25~-35°C, and the slice thickness is 18~25 microns.

The fourth step, the removal of non-specific fluorescence

Soak the slices in glycine solution for 30-40 minutes, then wash with PBS buffer 1-2 times, each time for 5 minutes, and the washing temperature is 28-30 degrees;

Step 5. Immunolabeling

a. Put the slices into the primary antibody working solution against α-tubulin, and incubate at 32°C for 2 hours at a time;

b. Rinse the slices after one incubation with washing solution three times, each time for at least 8 minutes;

c. Put the rinsed slices into FITC-labeled secondary antibody working solution, and incubate for a second time at 37°C in a dark environment for 2 hours;

d. Rinse the slices after the second incubation with the washing solution three times, at least 8 minutes each time;

The sixth step, sealing

The immunolabeled sections were sealed with PBS-glycerol solution, and the immunofluorescent antibody labeling of the microtubule skeleton of young cotton tissues was completed so far.

The technical solution of the present invention can also be realized in the following way: in the first step, in the fixative solution fixing, the described fixative solution is 0.4% glutaraldehyde containing mass percent, 3.6% paraformaldehyde, mass percent 0.02% Triton-100, pipes, MEGTA and MgSO4, wherein the molar concentration of pipes is 50 mM, the molar concentration of MEGTA is 5 mM, and the molar concentration of MgSO4 is 5 mM.

The technical solution of the present invention can also be realized in the following way: in the first step, in the fixation with the fixative, the young cotton tissue refers to the tissue 1 cm below the cotton stem tip, or the tissue 1 cm above the cotton root tip, cut out The length of the young cotton tissue is less than or equal to 3 insides, and the thickness is less than or equal to 1 cm.

The technical scheme of the present invention can also be realized in the following way: in the second step, during dehydration, first put the cotton tender tissues fixed by the fixative solution into 0.5% sucrose-phosphate buffer solution and soak for 1 to 3 hours , then soaked in a sucrose-phosphate buffer solution with a mass volume ratio of 1.5% for 1 to 3 hours, then soaked in a sucrose-phosphate buffer solution with a mass volume ratio of 3% for 1 to 3 hours, and finally put in a mass volume ratio of Soak in 4% sucrose-phosphate buffer for 1-3 hours.

The technical solution of the present invention can also be realized as follows: in the second step, during dehydration, the pH value of the sucrose-phosphate buffer solution is 7.1-7.5.

The technical solution of the present invention can also be realized in the following way: In the third step, when frozen sectioning, first immerse the dehydrated cotton tender tissue in the OCT embedding agent for 8-10 minutes, and then put the cotton tender tissue into freezing Freeze in the microtome at -25~-35°C for 10~15 minutes, and finally fix it for sectioning.

The technical solution of the present invention can also be realized as follows: in the fourth step, in the removal of non-specific fluorescence, the molar concentration of the glycine solution is 80 mM, and the pH value of the PBS buffer is 7.3.

The technical solution of the present invention can also be realized in the following way: in the fifth step, in the immunolabeling, both the first antibody working solution and the second antibody working solution contain 1% bovine serum albumin, and the first antibody working solution is Anti-αtubulin antibody solution diluted 200 times with PBS solution, and the secondary antibody working solution is FITC-labeled anti-αtubulin antibody solution diluted 100 times with PBS solution.

The technical solution of the present invention can also be realized in the following way: in the fifth step, in the immunolabeling, the washing solution is PBS buffer.

The technical scheme of the present invention can also be realized like this: in the 6th step, in the sealing, the volume ratio of PBS and glycerol in the described PBS-glycerin solution is 1:1, and m-phenylenediamine is contained in the PBS-glycerol solution, The mass volume ratio of m-phenylenediamine to the PBS-glycerol solution is 0.1:100, and the pH value of the PBS-glycerol solution is 8.5-9.0.

The beneficial effects of the present invention are:

1. The present invention is to combine fluorescein with the microtubule skeleton of the tender cotton part, carry out immunolabeling to the microtubule skeleton of the young tissue of cotton, observe the change of the microtubule skeleton of the young tissue from the tissue level, and detect the cotton microtubule skeleton. The resistance to abiotic stress and the resistance of cotton to diseases and insect pests are studied.

2. The present invention carries out gradient dehydration through 0.5%, 1.5%, 3%, 4% sucrose phosphate buffer solution to the young cotton tissue fixed by the fixative, and the young cotton tissue has a relatively large water content. The temperature gradually balances with the room temperature, and the water in the cells liquefies and expands, which easily bursts the tissue cells, leaving many hole-like gaps on the tissue slices, resulting in damage to the cell structure, and even the displacement of the microtubule structure, which affects the experiment. the accuracy of the results. Therefore, it is necessary to perform gradient dehydration steps on young cotton tissues, which can effectively alleviate this phenomenon.

3. The present invention adopts sucrose phosphate buffer solution to dehydrate young cotton tissues, and then freezes them into sections. At this time, there are sucrose residues on the surface of the tissues. When freezing, sucrose is used as a cryoprotectant, and sucrose can combine with the moisture in tissue cells. Lower the freezing point of the intracellular solution, reduce the formation of ice crystals or larger ice crystals, thereby reducing cell damage. The adoption of frozen section technology not only makes the experiment fast, simple, and easy to operate, but also shortens the slow and complicated process of conventional paraffin sectioning. During the treatment process, the damage to the active substances of the sample tissue is reduced, the activity of biomolecules is better preserved, and the cell wall is destroyed to a certain extent. Under freezing conditions, the cell wall is opened by mechanical force, and the shape of the cell will not change at the same time. Microtubule antibodies enter cells and bind to microtubules.

4. The fourth step of the present invention eliminates part of the non-specific fluorescence. Through the treatment with higher concentration of glycine, the interference of the autofluorescence of the residual aldehydes in the fixative to the experimental results is avoided, so that the fluorescent signal is stronger and the background The signal is weaker and the microtubule skeleton is clearer.

5. The steps of the present invention are simple, easy to master, clear results and high practical application value. It opens up a new direction for cotton cell biology research, and can also make up for the defects of cotton molecular biology research. The invention is not only applicable to cotton young parts, but also has certain reference value for the observation of microtubule skeletons in other young plant parts.

Description of drawings

Fig. 1 is the observation result of the microtubule skeleton of the outer cortex cells of the main stem at 1 cm below the stem tip of the three-leaf stage of cotton;

Fig. 2 is the observation result of the microtubule skeleton of the root cortex at 1 cm above the root tip of the cotton three-leaf stage;

Fig. 3 is the observation result of the microtubule skeleton in the outer cortex of the root at 0.5 cm above the root tip of cotton at the three-leaf stage.

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

Embodiment one

In this example, immunofluorescent labeling was performed on the microtubule skeleton of the outer cortex cells of the main stem 1 cm below the shoot tip of the cotton three-leaf stage.

A method for immunofluorescence antibody labeling of cotton young tissue microtubule skeleton, comprising the following steps:

The first step, fixative fixation

Cut the outer skin of the main stem 1 cm below the tip of the cotton three-leaf stage with bare hands, put the outer skin of the main stem into a sealed container filled with fixative, vacuumize the sealed container for 15 minutes, and then place the sealed container at 28°C For 3 hours, the fixative in the sealed container fixes the young cotton tissue;

Described stationary liquid is to contain the glutaraldehyde that mass percent is 0.4%, the paraformaldehyde that mass percent is 3.6%, the Triton-100 that mass percent is 0.02%, pipes, MEGTA and MgSO , wherein the molar concentration of pipes is 50 mM, the molar concentration of MEGTA is 5 mM, and the molar concentration of MgSO4 is 5 mM. The second step, dehydration

The cotton main stem cortex in the airtight container is taken out, and then the main stem cortex is put into sucrose phosphate buffer solution to soak and dehydrate; the pH value of the sucrose phosphate buffer solution is 7.3.

When dehydrating, put the cotton main stem cortex fixed by the fixative into 0.5% sucrose-phosphate buffer (PBS buffer) for 1 hour, and then put in 1.5% sucrose- Soak in phosphate buffer solution (PBS buffer solution) for 1 hour, then put it into 3% sucrose-phosphate buffer solution (PBS buffer solution) by mass volume ratio and soak it for 1 hour, and finally put it into 4% sucrose- Soak in phosphate buffered solution (PBS buffer) for 1 hour.

Step 3: Frozen Sectioning

First immerse the dehydrated cotton main stem cortex in OCT embedding agent for 10 minutes, then put the cotton main stem cortex into a cryostat and freeze it at -25°C for 15 minutes, and finally fix it and slice it with a thickness of 25 Microns.

The fourth step, the removal of non-specific fluorescence

Soak the slices in a glycine solution with a molar concentration of 80 mM for 40 minutes, then wash the slices twice with PBS buffer, 5 min each time, at a temperature of 28 degrees; the pH of the PBS buffer is 7.3.

Step 5. Immunolabeling

a. Put the slices into the primary antibody working solution against α-tubulin, and incubate at 32°C for 2 hours at a time; The solution contains 1% bovine serum albumin.

b. Rinse the sections after the first incubation with the washing solution for three times, 8 minutes each time; the washing solution is PBS buffer.

c. Put the rinsed slices into FITC-labeled secondary antibody working solution, and incubate for 2 hours in a dark environment at 37°C; the secondary antibody working solution is FITC-labeled anti- αtubulin antibody solution diluent and secondary antibody working solution both contain 1% bovine serum albumin.

d. Rinse the slices after the second incubation three times with the washing solution for 8 minutes each time; the washing solution is PBS buffer solution.

The sixth step, sealing

The immunolabeled sections were sealed with PBS-glycerol solution, and the immunofluorescent antibody labeling of the microtubule skeleton of young cotton tissues was completed so far.

The volume ratio of PBS and glycerol in the PBS-glycerin solution is 1:1, and the PBS-glycerol solution contains m-phenylenediamine, and the mass-volume ratio of m-phenylenediamine and PBS-glycerol solution is 0.1:100, and the The pH of the PBS-glycerol solution is 9.0.

The cotton main stem cortex slices after sealing were put into observation with a laser confocal microscope, and photographed, and the resulting photographs are shown in Figure 1.

It can be seen from Figure 1 that after the immunofluorescent labeling of the microtubule skeleton of the outer cortex cells of the main stem 1 cm below the shoot tip of the three-leaf stage of cotton, the distribution of the microtubule skeleton can be clearly observed under the laser confocal microscope.

Embodiment two

Repeat Example 1, with the following differences. In this example, the root cortex at 1 cm above the root tip of the cotton three-leaf stage is used for immunofluorescent antibody labeling. The specific implementation steps are as follows:

The first step, fixative fixation

Cut the root cortex at 1 cm above the root tip of cotton at the three-leaf stage with bare hands, put the root cortex into a sealed container filled with fixative, vacuumize the sealed container for 10 minutes, and then put the sealed container at 28 ° C for 2 Hours, the fixative in the airtight container fixes the tender tissue of cotton;

Described stationary liquid is to contain the glutaraldehyde that mass percent is 0.4%, the paraformaldehyde that mass percent is 3.6%, the Triton-100 that mass percent is 0.02%, pipes, MEGTA and MgSO , wherein the molar concentration of pipes is 50 mM, the molar concentration of MEGTA is 5 mM, and the molar concentration of MgSO4 is 5 mM. The second step, dehydration

The cotton root cortex in the airtight container is taken out, and then the root cortex is soaked in sucrose phosphate buffer solution for dehydration; the pH value of the sucrose phosphate buffer solution is 7.5.

When dehydrating, first put the cotton root cortex fixed by the fixative into 0.5% sucrose-phosphate buffer (PBS buffer) for 2 hours, and then put in 1.5% sucrose-phosphate Soak in buffer (PBS buffer) for 2 hours, then soak in sucrose-phosphate buffer (PBS buffer) with a mass volume ratio of 3% for 2 hours, and finally put in sucrose-phosphate buffer with a mass volume ratio of 4% buffer (PBS buffer) for 2 hours.

Step 3: Frozen Sectioning

First immerse the dehydrated cotton root cortex in the OCT embedding agent for 8 minutes, then put the cotton root cortex into a cryostat and freeze at -30°C for 15 minutes, and finally fix it and slice it with a thickness of 20 microns.

The fourth step, the removal of non-specific fluorescence

Soak the slices in a glycine solution with a molar concentration of 80 mM for 30 minutes, then wash the slices once with PBS buffer for 5 minutes at a temperature of 28 degrees; the pH of the PBS buffer is 7.3.

Step 5. Immunolabeling

a. Put the slices into the primary antibody working solution against α-tubulin, and incubate at 32°C for 2 hours at a time; The solution contains 1% bovine serum albumin.

b. Rinse the sections after the first incubation with the washing solution for three times, 8 minutes each time; the washing solution is PBS buffer.

c. Put the rinsed slices into FITC-labeled secondary antibody working solution, and incubate for 2 hours in a dark environment at 37°C; the secondary antibody working solution is FITC-labeled anti- αtubulin antibody solution diluent and secondary antibody working solution both contain 1% bovine serum albumin.

d. Rinse the slices after the second incubation three times with the washing solution for 8 minutes each time; the washing solution is PBS buffer solution.

The sixth step, sealing

The immunolabeled sections were sealed with PBS-glycerol solution, and the immunofluorescent antibody labeling of the microtubule skeleton of young cotton tissues was completed so far.

The volume ratio of PBS and glycerol in the PBS-glycerin solution is 1:1, and the PBS-glycerol solution contains m-phenylenediamine, and the mass-volume ratio of m-phenylenediamine and PBS-glycerol solution is 0.1:100, and the The pH of the PBS-glycerol solution is 8.5.

The cotton root cortex slices after sealing were put into observation with a laser confocal microscope, and photographed, the resulting photographs are shown in Figure 2,

It can be seen from Figure 2 that after the immunofluorescent labeling of the microtubule skeleton in the outer cortex cells of the root 1 cm above the root tip of cotton at the three-leaf stage, the distribution of the microtubule skeleton can be clearly observed under the laser confocal microscope.

Embodiment Three

Example 1 was repeated, with the following differences. In this example, immunofluorescent labeling was performed on the microtubule skeleton of the root epidermis at 0.5 cm above the root tip of cotton at the three-leaf stage.

A method for immunofluorescence antibody labeling of cotton young tissue microtubule skeleton, comprising the following steps:

The first step, fixative fixation

Cut the root cortex at 0.5 cm above the root tip of cotton at the three-leaf stage with bare hands, put the root cortex into a sealed container filled with fixative, vacuumize the sealed container for 8 minutes, and then place the sealed container at 30°C for 2 Hours, the fixative in the airtight container fixes the tender tissue of cotton;

Described stationary liquid is to contain the glutaraldehyde that mass percent is 0.4%, the paraformaldehyde that mass percent is 3.6%, the Triton-100 that mass percent is 0.02%, pipes, MEGTA and MgSO , wherein the molar concentration of pipes is 50 mM, the molar concentration of MEGTA is 5 mM, and the molar concentration of MgSO4 is 5 mM. The second step, dehydration

The cotton root cortex in the airtight container is taken out, and then the root cortex is soaked in sucrose phosphate buffer solution for dehydration; the pH value of the sucrose phosphate buffer solution is 7.3.

When dehydrating, first put the cotton root cortex fixed by the fixative into 0.5% sucrose-phosphate buffer (PBS buffer) for 2 hours, and then put in 1.5% sucrose-phosphate Soak in buffer (PBS buffer) for 2 hours, then soak in sucrose-phosphate buffer (PBS buffer) with a mass volume ratio of 3% for 2 hours, and finally put in sucrose-phosphate buffer with a mass volume ratio of 4% buffer (PBS buffer) for 2 hours.

Step 3: Frozen Sectioning

First immerse the dehydrated cotton main stem cortex in OCT embedding agent for 10 minutes, then put the cotton main stem cortex into a cryostat and freeze it at -35°C for 15 minutes, and finally slice it, with a thickness of 18 Microns.

The fourth step, the removal of non-specific fluorescence

Soak the slices in a glycine solution with a molar concentration of 80 mM for 30 minutes, then wash the slices twice with PBS buffer for 5 min at a temperature of 28 degrees; the pH of the PBS buffer is 7.3.

Step 5. Immunolabeling

a. Put the slices into the primary antibody working solution against α-tubulin, and incubate at 32°C for 2 hours at a time; The solution contains 1% bovine serum albumin.

b. Rinse the sections after the first incubation with the washing solution for three times, 8 minutes each time; the washing solution is PBS buffer.

c. Put the rinsed slices into FITC-labeled secondary antibody working solution, and incubate for 2 hours in a dark environment at 37°C; the secondary antibody working solution is FITC-labeled anti- αtubulin antibody solution diluent and secondary antibody working solution both contain 1% bovine serum albumin.

d. Rinse the slices after the second incubation three times with the washing solution for 8 minutes each time; the washing solution is PBS buffer solution.

The sixth step, sealing

The immunolabeled sections were sealed with PBS-glycerol solution, and the immunofluorescent antibody labeling of the microtubule skeleton of young cotton tissues was completed so far.

The volume ratio of PBS and glycerol in the PBS-glycerin solution is 1:1, and the PBS-glycerol solution contains m-phenylenediamine, and the mass-volume ratio of m-phenylenediamine and PBS-glycerol solution is 0.1:100, and the The pH of the PBS-glycerol solution is 9.0.

Put the cotton root cortex slices after sealing into the observation with a laser confocal microscope, and take pictures. The resulting pictures are shown in Figure 3.

It can be seen from Figure 3 that after immunofluorescent labeling of the microtubule skeleton in the root cortex at 0.5 cm above the root tip of cotton at the three-leaf stage, the distribution of the microtubule skeleton can be clearly observed under the laser confocal microscope.

It should be noted that the above-mentioned embodiments are illustrations rather than limitations to the technical solutions of the present invention, equivalent replacements by those of ordinary skill in the art or other modifications made according to the prior art, as long as they do not exceed the thinking and scope of the technical solutions of the present invention , should be included within the scope of the claims of the present invention.

Claims (10)

1. a kind of Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton, it is characterised in that：Including following technique Step：

The first step, fixer are fixed

Cotton tender tissue is cut, the tender tissue is put into the sealing container equipped with fixer, sealing container is vacuumized 10 ~ 15 minutes, sealing container is then stood 1.5 ~ 3 hours at a temperature of 25 ~ 30 DEG C, the fixer in sealing container is by cotton Tender tissue is fixed；

Second step, dehydration

Cotton tender tissue in sealing container is taken out, immersion is de- during the tender tissue then is put into sucrose phosphate buffer solution Water；

3rd step, freezing microtome section

Cotton tender tissue after dehydration is put into freezing-microtome and is cut into slices, freezing-microtome internal temperature is -25 ~ -35 DEG C, 18 ~ 25 microns of slice thickness,

4th step, the removal of non-specific fluorescence

Section is put into glycine solution and is soaked 30-40 minutes, then rinsed 1 ~ 2 time with PBS, each 5min, rinsed Temperature 28-30 degree；

It is 5th step, immune labeled

A, will section be put into the primary antibody working solution of anti alpha-tubulin, be incubated next time 2 hours in 32 DEG C of temperature；

B, by once be incubated after section rinsing liquid rinse repeatedly three times, every time at least 8 minutes；

C, the section after rinsing is put into FITC mark secondary antibody working solution in, it is secondary at a temperature of dark surrounds and 37 DEG C to incubate Educate 2 hours；

D, the section rinsing liquid after secondary incubation is rinsed repeatedly three times, every time at least 8 minutes；

6th step, mounting

Using PBS-glycerite will be immune labeled after section mounting, so far complete cotton tender tissue microtubule skeleton and exempt from Epidemic disease fluorescence antibody is marked.

2. the Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton according to claim 1, its feature exists In：During the first step, fixer are fixed, described fixer is to contain the glutaraldehyde, mass percent that mass percent is 0.4% Paraformaldehyde, Triton-100, pipes, MEGTA and MgSO4 that mass percent is 0.02% for 3.6%, wherein pipes's Molar concentration is 50 mM, and the molar concentration of MEGTA is 5 mM, and the molar concentration of MgSO4 is 5 mM.

3. the Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton according to claim 1, its feature exists In：During the first step, fixer are fixed, described cotton tender tissue refers to 1 centimetre below Shoot Apex of Cotton of tissue, or is Levant Cotton Root 1 centimetre of tissue more than sharp, less than or equal to 3 the insides, thickness is less than or equal to 1 centimetre to the length of the cotton tender tissue for cutting.

4. the Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton according to claim 1, its feature exists In：When second step, dehydration, the cotton tender tissue after first fixer is fixed is put into sucrose-phosphorus that mass volume ratio is 0.5% Soaked 1 ~ 3 hour in acid buffer, place into and soak 1 ~ 3 hour in sucrose-phosphate buffer that mass volume ratio is 1.5%, so It is put into afterwards in sucrose-phosphate buffer that mass volume ratio is 3% and soaks 1 ~ 3 hour, is finally putting into the sugarcane that mass volume ratio is 4% Soaked 1 ~ 3 hour in sugar-phosphate buffer.

5. the Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton according to claim 1, its feature exists In：When second step, dehydration, the pH value of described sucrose-phosphate buffer is 7.1 ~ 7.5.

6. the Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton according to claim 1, its feature exists In：When the 3rd step, freezing microtome section, first the cotton tender tissue after dehydration is immersed in 8 ~ 10 minutes in OCT embedding mediums, then will Cotton tender tissue is put into freezing-microtome and is freezed 10 ~ 15 minutes at a temperature of -25 ~ -35 DEG C, is finally fixed again and cuts Piece.

7. the Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton according to claim 1, its feature exists In：In 4th step, the removal of non-specific fluorescence, described glycine solution obtains molar concentration for 80 mM, and described PBS delays The pH value of fliud flushing is 7.3.

8. the Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton according to claim 1, its feature exists In：5th step, it is immune labeled in, in described primary antibody working solution and secondary antibody working solution contain 1% bovine serum albumin, it is described Primary antibody working solution is to dilute 200 times of anti-α tubulin antibody-solutions dilutions, described secondary antibody working solution through PBS solution It is the anti-α tubulin antibody-solutions dilutions of the FITC marks that 100 times are diluted through PBS solution.

9. the Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton according to claim 1, its feature exists In：5th step, it is immune labeled in, described rinsing liquid is PBS.

10. the Immunofluorescent Antibody labeling method of cotton tender tissue microtubule skeleton according to claim 1, its feature exists In：In 6th step, mounting, PBS and the volume ratio of glycerine are 1 in described PBS-glycerite:In 1, PBS-glycerite Containing m-phenylene diamine (MPD), m-phenylene diamine (MPD) is 0.1 with the mass volume ratio of PBS-glycerite：100, described PBS-glycerite PH value be 8.5 ~ 9.0.