CN113008648A - Fluorescence microscopy method for observing behavior of peony pollen tube by combining improved paraffin section with aniline blue dyeing method - Google Patents
Fluorescence microscopy method for observing behavior of peony pollen tube by combining improved paraffin section with aniline blue dyeing method Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 36
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 title claims abstract description 23
- 241000736199 Paeonia Species 0.000 title claims abstract description 22
- 235000006484 Paeonia officinalis Nutrition 0.000 title claims abstract description 21
- 239000012188 paraffin wax Substances 0.000 title claims abstract description 21
- 238000004043 dyeing Methods 0.000 title claims abstract description 13
- 238000000799 fluorescence microscopy Methods 0.000 title claims abstract description 6
- 239000001993 wax Substances 0.000 claims abstract description 26
- 239000000463 material Substances 0.000 claims abstract description 22
- 238000007447 staining method Methods 0.000 claims abstract 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 89
- 239000000243 solution Substances 0.000 claims description 42
- 235000019441 ethanol Nutrition 0.000 claims description 28
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 20
- 238000002791 soaking Methods 0.000 claims description 18
- 239000008096 xylene Substances 0.000 claims description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 14
- 239000011521 glass Substances 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 8
- 229960000583 acetic acid Drugs 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 239000012362 glacial acetic acid Substances 0.000 claims description 7
- 108010010803 Gelatin Proteins 0.000 claims description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000000853 adhesive Substances 0.000 claims description 6
- 230000001070 adhesive effect Effects 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 229920000159 gelatin Polymers 0.000 claims description 6
- 239000008273 gelatin Substances 0.000 claims description 6
- 235000019322 gelatine Nutrition 0.000 claims description 6
- 235000011852 gelatine desserts Nutrition 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 5
- 230000010152 pollination Effects 0.000 claims description 5
- 239000012024 dehydrating agents Substances 0.000 claims description 4
- 238000007598 dipping method Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 3
- 229910000404 tripotassium phosphate Inorganic materials 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims 2
- 230000003187 abdominal effect Effects 0.000 claims 1
- 238000007654 immersion Methods 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 239000001045 blue dye Substances 0.000 abstract 1
- 230000008595 infiltration Effects 0.000 abstract 1
- 238000001764 infiltration Methods 0.000 abstract 1
- 230000006399 behavior Effects 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 210000001672 ovary Anatomy 0.000 description 6
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- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 2
- 240000005001 Paeonia suffruticosa Species 0.000 description 1
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- 210000001161 mammalian embryo Anatomy 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000014639 sexual reproduction Effects 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
-
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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Abstract
The invention relates to the technical field of plant sample observation, and more particularly relates to a fluorescence microscopy method for observing peony pollen tube behavior by combining an improved paraffin section with an aniline blue staining method, wherein the method comprises the following steps: (1) dissecting and separating the carpel material of the peony to be observed; (2) fixing, dehydrating and transparent the separated material according to a modified paraffin slicing method which is not carried out in an oven overnight; (3) wax infiltration and embedding; (4) slicing, unfolding and unfolding; (5) dyeing with water-soluble aniline blue dye liquor; (6) and (5) carrying out fluorescence microscopic observation on the pollen tube. By adopting the technical scheme, the peony pollen tube behavior observation is more efficient than the traditional tabletting observation, and the observation effect is optimized.
Description
Technical Field
The invention relates to the technical field of plant microscopy, in particular to a fluorescence microscopy method for observing the behavior of a peony pollen tube by combining an improved paraffin section with an aniline blue dyeing method.
Background
After the peony pollinates, the pollen tube germinates on the stigma to form the pollen tube containing sperm cells and nutritional nuclei, the pollen tube descends along the way of the stigma, enters an embryo sac from a pearl hole in an ovary, and combines sperms and ova to complete fertilization, which is an important process for completing sexual reproduction of the peony.
So far, the observation of the growth process of the plant pollen tube mainly depends on tabletting and dyeing, firstly, the plant material is softened by NaOH solution, then the plant material is impregnated and dyed by aniline blue fuel, and then the tabletting is carried out for fluorescence observation. The ovule of peony is large, and the traditional tabletting can not observe the complete growth process of the pollen tube in the stylobate passage and the ovary, and can not obtain the specific time and shape of the pollen tube entering the ovule. In the traditional method, plant materials are only softened and are not transparent, so that the observation effect is influenced during microscopic examination, and the growth track and the development process of the pollen tube cannot be really observed. The liquid paraffin used in the tabletting process is easy to solidify and can be uniformly mixed with the dimethylbenzene for a long time; the oven needs to be used overnight, which causes certain hidden trouble to the laboratory safety.
The invention aims to solve the technical problem that the defect that comprehensive pollen tube behaviors and complete information of a fertilization process cannot be obtained due to the fact that a peony ovary wall to be measured is thick and observation is difficult or an ovule is large and has to be divided in the existing post-pollination fluorescent microscopic tabletting observation method is overcome, and the fluorescent microscopic observation method for the peony post-pollination pollen tube behaviors is safe in a flaking process, good in flaking effect and clear and visible in the pollen tube and fertilization process.
Disclosure of Invention
The invention provides a fluorescence microscopic observation method for pollen tube behaviors after peony pollination. The method comprises the steps of sampling and fixing a material to be detected, dehydrating and transparentizing, wax dipping and embedding, slicing, sticking and spreading, dyeing, observing and the like, and the specific operation of each step is as follows:
the first step is as follows:
material fixation: taking off the carpel of pollinated peony to be observed as a material to be detected, separating the stigma from a long and narrow band along a dorsal suture and a ventral suture under a dissecting mirror into two parts by using a scalpel, immersing the two parts into Carnot stationary liquid, placing the two parts into a vacuum drier, settling the material, transferring the material into an ethanol solution with the volume fraction of 70% after 12 hours, and storing the material at 4 ℃.
The preparation method of the carnot stationary liquid comprises the following steps: mixing 95% ethanol water solution and glacial acetic acid according to the volume ratio of 3: 1.
The second step is that:
dehydrating and transparent: pouring out the total amount of liquid for fixing the sample, and replacing the dehydrating agent and the clearing agent with different gradients therein, comprising the following steps: soaking in 85% ethanol solution for 2 hr, soaking in 95% ethanol solution for 2 hr, and soaking in anhydrous ethanol solution for 2 times, each for 1.5 hr; soaking the mixture of absolute ethyl alcohol and xylene in a volume ratio of 1:1 for 2 hours, and soaking the mixture of the transparent agent and the pure xylene for 2 times, wherein each time lasts for 1.5 hours.
The third step:
wax dipping and embedding: pouring out the pure xylene, adding crude wax powder, adding the pure xylene with the same volume as the crude wax powder, placing in a constant temperature box at 60 ℃ for 2 hours without opening a cover, and closing the constant temperature box after the crude wax powder is completely melted; the next day, the cover was opened and placed in a 60 ℃ incubator for 4 hours without opening the cover, and then the cover was opened for 4 hours. The pure paraffin solution was then displaced 3 times: the interval time of the first wax replacement is 4 hours, the interval time of the second wax replacement and the third wax replacement is 2 hours, and after the paraffin solution is replaced for the last time for 2 hours, the sample is taken out from the paraffin solution and placed in an embedding box for embedding.
The fourth step:
slicing, sticking and unfolding: the obtained wax block was trimmed to a regular trapezoid shape, fixed in an embedding cassette, and sliced on a slicer to a slice thickness of 14 μm. Spreading the cut wax tape on a glass slide coated with a Hebert adhesive, and heating the glass slide with the wax tape on a 40 ℃ spreading machine until the glass slide is completely stretched.
The preparation method of the Heubart adhesive comprises the following steps: liquid A: 1g of gelatin is put into 100ml of distilled water, and 15ml of glycerol and 2g of phenol are added after the gelatin is completely dissolved at the constant temperature of 40 ℃; b, liquid B: 4ml of formaldehyde and 100ml of distilled water are firstly dripped with 1 drop of the liquid A when in use, and then dripped with 2-3 drops of the liquid B after being evenly coated on a glass slide and evenly spread.
The fifth step:
and (3) aniline blue dyeing: sequentially soaking with xylene for 2 times, anhydrous ethanol and xylene mixed solution (1:1V/V), anhydrous ethanol for 2 times, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, and 30% ethanol for 3min each time, rinsing in buffer solution with pH of 6.7 for 20min, and dyeing in 0.1% aniline blue solution for 3 hr.
preparation method of pH 6.7 buffer solution：1mol.L-1The NaOH solution is mixed with 45 percent glacial acetic acid, the pH value is adjusted to 6.7, and the mixture is stored at normal temperature.
The preparation method of the 0.1 percent aniline blue solution comprises the following steps: 0.1g aniline blue, 0.71g K3PO4The volume of distilled water is fixed to 100ml, the pH value is adjusted to 8.5, and the mixture is stored in a dark place.
And a sixth step:
and (3) observation by a fluorescence microscope: after being dyed by 0.1 percent aniline blue solution, the film is not sealed and is directly placed under a fluorescence microscope for observation and photographing.
Compared with the prior art, the invention has the beneficial effects that:
the peony ovule is large, the traditional tabletting observation cannot obtain the whole growth process track of a complete pollen tube in a stylar tract and an ovary, and the specific time and shape of the pollen tube entering the ovule cannot be observed. By adopting the technical scheme of the invention, oven equipment is not needed to be used overnight, and the operation is safer than that of the traditional paraffin slicing method. The experimental effect chart proves that by adopting the technical scheme of the invention, the continuous behavior tracks of the pollen tube in the styloid tract and the ovary can be observed, the ovule is clear and visible, the fertilization information is comprehensive and complete, and reliable data and technical support can be provided for objectively and scientifically analyzing the behavior characteristics of the peony pollen tube.
Drawings
FIG. 1 shows a peony bark tablet made by a conventional whole ovary tabletting method. In FIG. 1, Ov denotes an ovule and PT denotes a pollen tube.
FIG. 2 is a section of a large yellow peony pollination boll channel manufactured by the method. PT in FIG. 2 shows the pollen tube.
FIG. 3 is the observation of the ovule section of the peony pollen made by the method. The ovule shown in FIG. 3 is the ovule where the pollen tube enters the fertilization, Ov indicates the ovule, and PT indicates the pollen tube.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1 Observation of pollen tube behavior after pollination of Paeonia macrocarpa
The first step is as follows: taking pistil carpel as a material to be detected after pollination of Paeonia suffruticosa, separating a long and narrow band of a stigma along a dorsal suture and a ventral suture under a dissecting mirror into two parts by using a scalpel, immersing the two parts into Carnot stationary liquid, placing the two parts into a vacuum drier, settling the material to the bottom of the container, transferring the material into an ethanol solution with the volume fraction of 70% after 12 hours, and storing the material at 4 ℃.
The preparation method of the Noo stationary liquid comprises the following steps: the volume fraction is 95% ethanol water solution and glacial acetic acid mixed solution (3:1, V/V).
The second step is that: dehydrating and transparent: pouring out all liquid for fixing the sample, and replacing the dehydrating agent and the clearing agent with different gradients in the container, wherein the method comprises the following steps: soaking in 85% ethanol solution for 2 hr, soaking in 95% ethanol solution for 2 hr, and soaking in anhydrous ethanol solution for 2 times, each for 1.5 hr; soaking the mixture of absolute ethyl alcohol and xylene in a volume ratio of 1:1 for 2 hours, and soaking the mixture of the transparent agent and the pure xylene for 2 times, wherein each time lasts for 1.5 hours.
The third step: wax dipping and embedding: pouring out the pure xylene, adding crude wax powder, adding the pure xylene with the same volume as the crude wax powder, placing in a constant temperature box at 60 ℃ for 2 hours without opening a cover, and closing the constant temperature box after the paraffin wax powder is completely melted; the next day, the cover was opened and placed in a 60 ℃ incubator for 4 hours without opening the cover, and then the cover was opened for 4 hours. The pure paraffin solution was then displaced 3 times: the interval time of the first wax replacement is 4 hours, the interval time of the second wax replacement and the third wax replacement is 2 hours, and after the pure paraffin solution is replaced for the last time for 2 hours, the sample is taken out from the paraffin solution and placed in an embedding box for embedding.
The fourth step: slicing, sticking and unfolding: and trimming the wax block into a regular trapezoid shape and fixing the wax block in an embedding box. The sheet was cut into pieces with a thickness of 14 μm by a microtome. Spreading the cut wax tape on a glass slide coated with Hebert adhesive, and heating the glass slide on a 40 deg.C developing machine until the glass slide is completely stretched.
The preparation method of the Heubart adhesive comprises the following steps: liquid A: 1g of gelatin is put into 100ml of distilled water, and 15ml of glycerol and 2g of phenol are added after the gelatin is completely dissolved at the constant temperature of 40 ℃; b, liquid B: 4ml of formaldehyde and 100ml of distilled water are used, 1 drop of the first solution is firstly dripped, 2-3 drops of the second solution are dripped after the first solution is evenly coated on a glass slide, and the second solution is evenly spread.
The fifth step: and (3) aniline blue dyeing: sequentially soaking with xylene for 2 times, anhydrous ethanol and xylene mixed solution (1:1v/v), anhydrous ethanol for 2 times, 95% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, and 30% ethanol for 3min each time, rinsing in buffer solution with pH of 6.7 for 20min, and dyeing in 0.1% aniline blue solution for 3 h.
preparation method of pH 6.7 buffer solution: 1mol.L-1The NaOH solution is mixed with 45 percent glacial acetic acid, the pH value is adjusted to 6.7, and the mixture is stored at normal temperature.
The preparation method of the 0.1 percent aniline blue solution comprises the following steps: 0.1g aniline blue, 0.71g K3PO4The volume of distilled water is fixed to 100ml, the pH value is adjusted to 8.5, and the mixture is stored in a dark place.
And a sixth step: and (3) observation by a fluorescence microscope: after being dyed by 0.1 percent aniline blue solution, the film is not sealed and is directly placed under a fluorescence microscope for observation and photographing.
Claims (8)
1. A fluorescence microscopy method for observing a peony pollen tube by combining an improved paraffin section with an aniline blue staining method is characterized by comprising the following steps: (1) taking materials and fixing; (2) dehydrating and transparent; (3) wax dipping and embedding; (4) slicing, sticking and spreading; (5) aniline blue dyeing; (6) and (4) observing by a fluorescence microscope.
2. The method of claim 1, wherein the drawing material fixation comprises: taking the carpel of the peony to be observed after pollination as a material to be detected, dissecting and separating the carpel material of the peony to be observed from a long and narrow band of a stigma by using a scalpel under a dissecting mirror along a back suture line and an abdominal suture line, dividing the carpel material into two parts, immersing the separated material into Carnot fixing solution, placing the separated material into a vacuum drier to enable the sample to sink, transferring the sample into an ethanol solution with the volume fraction of 70% after 12 hours, and storing the sample at the low temperature of 4 ℃;
the Carnot stationary liquid comprises 95% ethanol aqueous solution and glacial acetic acid by volume fraction, and the volume ratio of the 95% ethanol aqueous solution to the glacial acetic acid is 3: 1.
3. The method of claim 1, wherein the dehydrating transparent comprises: and pouring out the total amount of the liquid for fixing the sample, and replacing the dehydrating agent and the clearing agent with different gradients therein for dehydrating and clearing.
4. The method according to claim 3, wherein the displacing different gradients of dehydrating agent and clearing agent for dehydrating and clearing comprises: soaking in 85% ethanol solution for 2 hr, soaking in 95% ethanol solution for 2 hr, and soaking in 100% ethanol solution for 2 times, each for 1.5 hr; soaking with 100% ethanol and xylene mixed solution at a volume ratio of 1:1 for 2h, and soaking with pure xylene for 2 times, each time for 1.5 h.
5. The method of claim 1, wherein the wax immersion embedding comprises: pouring out a transparent agent of pure xylene, adding crude wax powder, adding the pure xylene with the same volume, placing in a constant temperature box at 60 ℃ for 2 hours without opening a cover, and closing the constant temperature box after the paraffin powder is completely melted; and on the next day, the sample is placed in a 60 ℃ thermostat for 4 hours without opening the cover, the 60 ℃ thermostat is opened for 4 hours, then the pure paraffin solution is replaced by 3 times, the interval time of the first paraffin replacement is 4 hours, the interval time of the second paraffin replacement and the third paraffin replacement is 2 hours, and after the pure paraffin solution is replaced for 2 hours for the last time, the sample is taken out of the paraffin solution and placed in an embedding box for embedding.
6. The method of claim 1, wherein the slicing, sticking and spreading comprises: the obtained wax block was trimmed to a regular trapezoid shape, fixed to an embedding cassette, and placed on a microtome for sectioning, the section thickness being 14 μm. Slightly flattening the cut wax tape on a glass slide coated with a Hebert adhesive, and heating the glass slide on a 40 ℃ glass developing machine until the glass slide is completely stretched;
the Hehbert adhesive comprises: liquid A and liquid B, the liquid A is: 1g of gelatin is put into 100ml of distilled water, and 15ml of glycerol and 2g of phenol are added after the gelatin is completely dissolved at the constant temperature of 40 ℃; the liquid B comprises: 4ml of formaldehyde and 100ml of distilled water are firstly dripped with 1 drop of the liquid A when in use, and then dripped with 2-3 drops of the liquid B after being evenly coated on a glass slide for even paving.
7. The method of claim 1, wherein the aniline blue dyeing comprises: sequentially soaking xylene, a mixed solution of absolute ethyl alcohol and xylene in a volume ratio of 1:1, absolute ethyl alcohol, 95% ethyl alcohol, 85% ethyl alcohol, 70% ethyl alcohol, 50% ethyl alcohol and 30% ethyl alcohol for 3min, rinsing in a buffer solution with the pH value of 6.7 for 20min, and finally placing in a 0.1% aniline blue solution for dyeing for 3 h;
the buffer solution with pH 6.7 comprises: 1mol.L-1Mixing the NaOH solution with 45% glacial acetic acid, adjusting the pH value to 6.7, and storing at normal temperature;
the 0.1% aniline blue solution comprises: 0.1g aniline blue, 0.71g K3PO4The volume of distilled water is fixed to 100ml, the pH value is adjusted to 8.5, and the mixture is stored in a dark place.
8. The method of claim 1, wherein the fluorescence microscopy observation comprises: after being dyed by 0.1 percent aniline blue solution, the film is not sealed and is directly placed under a fluorescence microscope for observation and photographing.
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