CN110793829A - Method for slicing highland barley stem profile paraffin suitable for plateau environment - Google Patents
Method for slicing highland barley stem profile paraffin suitable for plateau environment Download PDFInfo
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
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- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 claims description 4
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- 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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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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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
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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Abstract
The invention discloses a method for slicing highland barley stem profile paraffin suitable for plateau environment, which comprises six steps of material taking and fixing, dehydration and transparency, waxing and embedding, slicing and unfolding, dewaxing and dyeing, mounting and observing. Because the tissue structure of the highland barley stalks is thin and crisp, the selection of dehydration time, wax immersion time and paraffin melting point is improved in the paraffin slicing process, and the complete paraffin slicing method of the highland barley stalks can be obtained. In the aspect of paraffin melting point, through continuous search and repeated tests, the paraffin with high melting point is used for soaking wax in the technology, and finally a whole set of paraffin slicing technology suitable for highland barley stem profiles is worked out, so that the technology has important significance for researching and understanding highland barley stem tissue structures and researching lodging-resistant mechanism in highland barley scientific research and production. The method realizes the fast production of the paraffin section, shortens the production time of the complete section by 1 to 4 days compared with the traditional paraffin section method, and saves the time on the basis of ensuring the section production effect.
Description
Technical Field
The invention belongs to the technical field of paraffin section of plant sample tissue, particularly relates to a paraffin section method of plant tissue, and particularly relates to a section paraffin section method of highland barley stalks suitable for plateau environment.
Background
Highland barley (Hordeum vulgare L.var. nudum hook.f.), belongs to the genus Hordeum gramineae, belongs to a variety of cultivated barley in botany, is called naked barley because the husk is separated from the caryopsis inside and outside the kernel and the kernel is naked, and is called highland barley in the Tibet plateau and is the most distinctive crop in the Tibet plateau. The highland barley grains have higher nutrition and health care functions and are widely used in wine brewing and processing industries, and the straws are also high-quality forage grass and have great effects in the development of animal husbandry. The highland barley industry has great significance for grain safety and regional economic development of the Tibetan region. The highland barley is shallower than the root system of the common wheat, and the stem is thin and soft, so the environmental climate condition of the planting area is poorer, and the highland barley is easy to fall down in the mature period. Lodging can cause severe yield loss of the highland barley and remarkable reduction of the quality of grains and forage grass, is not beneficial to mechanized harvesting, increases the manual investment and reduces the economic benefit. The lodging can disturb the spatial distribution structure of the leaves of the crops, so that the ventilation and light transmission conditions are worsened, the lower leaves are withered, yellow and rotten, the photosynthetic rate is sharply reduced, the synthesis of the assimilates is blocked, the growth and development of the plants are severely limited, and finally the yield of the crops is reduced and the quality is greatly reduced. The stalks are tissues and organs which support the weight of the plants and are directly acted by external environments such as wind, rain and the like, and the characteristics of the stalks are closely related to the lodging resistance of the highland barley. In order to solve the lodging problem in highland barley production and understand the lodging resistance of highland barley, a large amount of researches on stem external characteristics such as the height, internode length and straw thickness of highland barley straws are changed in different ways, and no relevant report is provided for the research on the internal tissue structure of highland barley straws.
Paraffin sectioning is one of the most important and commonly used methods in microscopy, and was cited by Klebs in 1869, primarily for the observation of cell tissue and organ morphology, due to its ability to slice into thin, continuous sections. This method is called paraffin sectioning, in which a material is embedded in paraffin and then sectioned together with the paraffin on a microtome. In the initial paraffin sectioning, the sectioned material was only surrounded by paraffin and was not infiltrated by paraffin. Paraffin embedding was not widely used until after a report on paraffin embedding technology was published by Bourne in 1882, plays an important role in the history of plant cell and tissue research, and will continue to play a role as a general technology in the future.
The traditional paraffin section method comprises the steps of material taking, fixing (12-24 hours in use), dehydration (9 steps, 11 hours in time), transparency (4 steps, 4-16 hours in time), wax dipping (16-24 hours in time), embedding, block trimming, section cutting, sheet spreading, sheet baking, dewaxing (50min), rehydration, dyeing, dehydration, transparency (22 steps, 3.5-14 hours in time), sheet sealing and drying and solidification. The traditional paraffin section method has complicated steps, and the section making effect can be influenced if one step of careless error occurs in the middle; the film-making cycle is long, and the film-making efficiency is low.
In recent years, many studies have been made on improvement of the method for producing paraffin sections. Zhoutonhua introduces a microwave water-bath radiation rapid paraffin section method, and obtains satisfactory effect in pathological diagnosis in operation. Weixin et al observe rice leaf tissue by microwave rapid paraffin sectioning, and the results show that the method can greatly shorten the treatment time and improve the working efficiency. The process of embedding and repairing the paraffin section of the plant tissue is improved by Jiangfujian and the like, so that the working efficiency is improved, the repaired paraffin blocks are neat and uniform, and the subsequent slicing process is not influenced. But the section paraffin section method of the highland barley stalks suitable for the plateau environment is very few.
The highland barley is a special grain and feed dual-purpose crop of the Qinghai-Tibet plateau, the tissue structure of the stem of the highland barley is thin and crisp, but no related literature can be used for reference, and in addition, the difference between the plateau environment and the inland is added, so that the paraffin section method and the crop lodging-resistant research of other crop tissues are consulted on the basis of the manual garlic chive tissue paraffin section copying technical method, repeated tests and groping are carried out, the second section of the base part of the highland barley is determined to be adopted for analysis, and the section paraffin section method of the stem of the highland barley suitable for the plateau environment is summarized by improving and adjusting the dehydration time, the wax immersion time, the selection of the paraffin melting point, the groping of the sheet expanding method, the dyeing time adjustment and the like, so that the method has important significance for researching and understanding the tissue structure of the stem of the highland barley and researching the lodging-resistant mechanism.
Based on the analysis, the preparation of the stationary liquid → material drawing → vacuum pumping → storage → washing → dehydration → transparence → wax soaking → embedding → slicing → exhibition sheet → dewaxing → dyeing → sealing sheet → observation is summarized, and a whole set of paraffin slicing technology suitable for highland barley stalk section is provided. Has important significance for researching and understanding the tissue structure of the stem of the highland barley and researching the lodging resistant mechanism in the scientific research and production of the highland barley.
Disclosure of Invention
Based on the analysis, the invention provides the highland barley stem profile paraffin slicing method suitable for the highland barley stem dehydration time and the paraffin immersion time, which adopts high-melting-point paraffin for paraffin immersion, has short dyeing time, saves time on the basis of ensuring the sheet making effect and is suitable for the plateau environment. The invention is realized by the following means:
a highland barley stem profile paraffin section method suitable for plateau environment comprises the steps of material taking and fixing, dehydration and transparency, wax dipping and embedding, section and slide unfolding, dewaxing and dyeing, slide sealing and observation.
Further, the material taking and fixing method comprises the following steps: taking the second internode of the basal part of the highland barley stalk of 2-4cm, quickly immersing the highland barley stalk into FAA fixing solution, vacuumizing and standing the highland barley stalk to obtain a highland barley stalk sample, and then fixing the highland barley stalk sample in a refrigerator at 4 ℃ for more than 24 hours.
Further, the dehydration and transparency method comprises the following steps: washing fixed highland barley stalks with 50% ethanol for 3 times, 1h each time, treating the washed highland barley internodes with 50% ethanol, 70% ethanol, 80% ethanol and 95% ethanol for 1h respectively, then transferring to absolute ethanol for 1.5h, and finishing dehydration; and (3) sequentially placing the dehydrated highland barley material into absolute ethyl alcohol, mixed solution of xylene and absolute ethyl alcohol which are mixed in equal volume, pure xylene and pure xylene for treatment, wherein the treatment in each step is carried out for 1-2 hours, so that the material is transparent.
Further, the wax dipping and embedding method comprises the following steps: selecting paraffin with melting point of 58-60 deg.C, dissolving and filtering, transferring transparent highland barley internode into mixed liquid incubator containing xylene and paraffin, and maintaining temperature, adding paraffin for several times; the material after being soaked in the wax is placed in the center of the two ends of the embedding box, and the embedding box is slowly placed into cold water to enable the paraffin to be rapidly solidified.
Further, the slicing and unfolding method comprises the following steps: fixing the embedded material on a slicer, manually shaking a pulley of the slicer, adjusting the slice thickness to 10-20 μm, cutting a complete wax tape, putting the cut wax tape into water at 40 ℃ to completely unfold the wax tape, inserting a glass slide below the wax tape in a water tank at 40-45 ℃, slightly fishing the wax tape, uniformly and flatly spreading the complete tissue on the glass slide, slowly dripping a sticky tablet between the glass slide and the wax tape, and air-drying in a natural environment to obtain a dry slice.
Further, the dewaxing and dyeing method comprises the following steps: respectively placing the dried slices into dimethylbenzene, mixed solution of dimethylbenzene and absolute ethyl alcohol which are mixed in equal volume, absolute ethyl alcohol, ethyl alcohol with the mass fraction of 95%, ethyl alcohol with the mass fraction of 80%, ethyl alcohol with the mass fraction of 70% and ethyl alcohol with the mass fraction of 50% for treatment for 5min until dewaxing completely; and then placing the dewaxed slices into a 1% safranin aqueous solution for dyeing for 30min, then placing the dyed slices into a 1% fast green solution for dyeing for 5s, and then sequentially placing the slices into 95% ethanol, absolute ethanol, a mixed solution of xylene and absolute ethanol, and xylene for 3min respectively to obtain the dyed slices.
Further, the mounting and observation method is as follows: selecting a cover glass with the thickness of 18mm multiplied by 18mm, and sealing the cover glass by using neutral gum; the images were taken significantly under 10 and 40 times using a BA210 biomicroscope manufactured by Miaodi, and the thick wall, the thickness and area of mechanical tissue, and the number and area of vascular bundles of the stem were measured using corresponding morphological analysis software.
Furthermore, the FAA fixing solution is prepared by a method comprising the following steps of taking 84 parts by volume of 70% alcohol, 5 parts by volume of formaldehyde, 5 parts by volume of glacial acetic acid (glacial acetic acid) and 6 parts by volume of glycerol, and uniformly mixing to obtain the FAA fixing solution for later use.
Further, the mixed solution of the absolute ethyl alcohol and the xylene is prepared by mixing 1 volume ratio of the absolute ethyl alcohol and 1 volume ratio of the xylene; the processing time of the dehydrated highland barley material in absolute ethyl alcohol, mixed solution of xylene and absolute ethyl alcohol with the same volume, pure xylene and pure xylene is 1-2 h.
Further, the temperature is kept at 58-62 ℃ in the incubator for 24h, pure paraffin is added every 6h, and the paraffin addition is completed in 3 times.
Further, the sticky tablet is prepared by the following method: mixing gelatin 1g, distilled water 100ml, and glycerol 15ml to obtain sticky tablet.
The invention has the beneficial effects that:
1. according to the characteristics of the highland barley stalks and the characteristics of the stalk tissue, repeated tests and searches are carried out to determine that the second internode of the highland barley base is adopted for analysis, and a whole set of paraffin section technology suitable for highland barley stalk sections in plateau environment is finally prepared by improving and adjusting the dehydration time, the wax immersion time, the selection of paraffin melting point, the search of a sheet spreading method and the adjustment of dyeing time, so that the method has important significance for researching and understanding the highland barley stalk tissue structure and researching lodging-resistant mechanism in scientific research and production of highland barley.
2. The method realizes the fast production of the paraffin section, shortens the production time of the complete section by 1 to 4 days compared with the traditional paraffin section method, and saves the time on the basis of ensuring the section production effect.
3. The paraffin section prepared by the invention can effectively avoid the deterioration of highland barley tissues and can be stored for a longer time. Secondly, the phenomenon of tissue shedding in the dewaxing process can be effectively prevented by adding the sticking agent. The section paraffin section of the highland barley stalk section obtained after adjustment is complete and continuous, has no defect, no overlap and distinct layers, and the highland barley epidermal cells are smooth and complete, and can clearly and accurately count and analyze the number of vascular bundles.
Drawings
FIG. 1 is a 10-fold under-the-mirror view of the cross-sectional structure of the highland barley stalks observed in the slicing method of example 1.
FIG. 2 is a 40-fold under-mirror view of the cross-sectional structure of the highland barley stalks observed under the slicing method of example 1.
FIG. 3 and FIG. 4 are 10-fold structural diagrams of the highland barley stalk section observed in the slicing method of comparative example 1.
Detailed Description
The following description of the embodiments of the present invention is provided in conjunction with the accompanying drawings of FIGS. 1-4:
example 1
(1) Preparing FAA stationary liquid: preparing a fixing solution by using 70% alcohol: formaldehyde: glacial acetic acid (glacial acetic acid): glycerol 84:5: 6;
(2) material taking: the second internode (2-4cm) of the highland barley stalk is quickly and completely immersed in the fixing solution;
(3) vacuumizing: 4 h;
(4) and (3) storage: placing the highland barley stalk sample completely immersed in the fixing solution and vacuumized in a refrigerator at 4 ℃ for more than 24 hours;
(5) washing: washing the FAA-fixed highland barley internodes with 50% ethanol for 3 times, one hour each time, thereby washing the FAA-fixed liquid in the material;
(6) and (3) dehydrating: sequentially using 50% ethanol, 70% ethanol, 80% ethanol, 95% ethanol for 1 hr, and anhydrous ethanol (90min) to ensure water to be completely removed;
(7) and (3) transparency: sequentially placing the dehydrated highland barley material into absolute ethyl alcohol, mixed solution of xylene and absolute ethyl alcohol which are mixed in equal volume, pure xylene and pure xylene for treatment, wherein the treatment in each step is carried out for 1-2 hours to ensure that the material is transparent;
(8) wax dipping: selecting paraffin with a melting point of 58-60 ℃, transferring highland barley internodes into a mixed liquid incubator filled with xylene and paraffin, keeping the incubator at a temperature of 58-62 ℃ (4-5 ℃ higher than the melting point of the paraffin), and keeping the temperature for 24h, wherein the paraffin is filtered once every 6h, and the paraffin addition is completed for 3 times so that the xylene in the material is slowly replaced by the paraffin;
(9) embedding: putting the material after being soaked in the wax into the center of two ends of an embedding box, slowly putting the embedding box into cold water, so that the paraffin is slowly solidified into a solid, paying attention to no need of twisting during embedding, and avoiding the phenomenon that a gap is formed between the paraffin and the material to generate bubbles and influence the slicing effect;
(10) slicing: the embedded material was fixed on a microtome (full automatic paraffin microtome ERM4000), the microtome was hand-operated with a pulley, the slice thickness was adjusted to 10-20 μm, a complete wax band was cut, and attention was paid to: the position of the blade can be properly adjusted (or the blade can be replaced in time) in the slicing process to ensure the slicing effect;
(11) exhibition of slices: putting the cut wax tape into warm water (40 ℃) to completely unfold the wax tape, inserting a glass slide into a water tank at an angle of 40-45 ℃ below the wax tape, slightly taking the wax tape out, uniformly and flatly paving the complete tissue on the glass slide, slowly dripping a sticky tablet (1 g of gelatin, 100ml of distilled water and 15ml of glycerol) along the space between the glass slide and the wax tape, and airing the glass slide in a natural environment;
(12) dewaxing: sequentially placing the glass slide with the wax band into mixed solution of xylene (until the wax band around the tissue completely falls off), equal volume of xylene and anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and 50% ethanol, and continuing dewaxing after repeating above process if complete dewaxing is not completed;
(13) dyeing: putting the dewaxed material into 1% safranin water solution for 30min, then sequentially putting the material into 50% ethanol, 70% ethanol and 80% ethanol for 3min respectively, putting the material into 1% fast green solution for dyeing for 5s, and sequentially putting the material into 95% ethanol, anhydrous ethanol, mixed solution of xylene and anhydrous ethanol with equal volume, xylene and xylene for 3min respectively;
(14) sealing: selecting a cover glass with the thickness of 18mm multiplied by 18mm, and sealing the cover glass by using neutral gum;
(15) and (4) observation: microscopic measurement the prepared paraffin section of the highland barley stalk is obviously shot under 10 and 40 times by using a BA210 biological microscope manufactured by Miaodi company, thick wall, mechanical tissue thickness and area, and vascular bundle quantity and area of the stalk are measured by using corresponding morphological analysis software, and the cross-sectional structure of the stalk is shown in figure 1 and figure 2.
Comparative example 1
(1) Preparing FAA stationary liquid: preparing a fixing solution by using 70% alcohol: formaldehyde: glacial acetic acid (glacial acetic acid): glycerol-84: 5:5: 6;
(2) material taking: the second internode (2-4cm) of the highland barley stalk is quickly and completely immersed in the fixing solution;
(3) washing: washing the fixed FAA internode with 50% ethanol for 3 times, and washing the FAA fixing solution in the material;
(4) and (3) dehydrating: sequentially and respectively dehydrating with different concentration gradients, dehydrating with 30% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, 95% ethanol, and 100% ethanol, and properly prolonging time in 100% ethanol to ensure that water is completely removed;
(5) and (3) transparency: placing the internodes in 100% ethanol, mixed solution of ethanol and xylene (volume ratio 1:1) in xylene in sequence to make the material transparent, and replacing the ethanol in the material with xylene;
(6) wax dipping: transferring the internode into a mixed liquid incubator filled with dimethylbenzene and paraffin, and adding paraffin (with a low melting point) to ensure that the paraffin in the material slowly enters the material; the wax dipping is put in a warm box, the temperature is 38 ℃ to 40 ℃, pure wax is gradually added for three times, and the paraffin wax slowly enters the material;
(7) embedding: the internode and fusion paraffin were poured into the embedding cassette so that the paraffin slowly solidified into a solid. The material should be placed in the center of the wax block;
(8) slicing: fixing the wax block with the material on a slicer, manually shaking a pulley of the slicer, and adjusting the thickness of the slice to cut a complete wax belt;
(9) exhibition of slices: putting the cut wax tape into warm water to completely unfold the wax tape, and then putting the wax tape on a glass slide for gluing; drying the water on the glass slide in an oven at 40 ℃;
(10) dewaxing: sequentially putting the glass slide with the wax band into a mixed solution of dimethylbenzene and ethanol (the volume ratio is 1:1), absolute ethyl alcohol, 95% ethanol, 80% ethanol, 70% ethanol and 50% ethanol, and removing paraffin in the material;
(11) dyeing: placing the slices into 1% safranine water solution, sequentially placing the slices into 50% ethanol, 70% ethanol, 80% ethanol, 1% fast green solution, dyeing for 30s, 95% ethanol, 100% ethanol, xylene mixed solution (1:1), xylene, and xylene for 3min respectively;
(12) sealing: a18 mm by 18mm coverslip was selected and mounted with neutral gum. The cross-sectional structure of the stalk is shown in fig. 3 and 4.
As can be seen from FIGS. 3 and 4, in the paraffin section technique search, the section of the stem of the highland barley obtained by the traditional or recorded method has rough cells of the epidermis of the highland barley, which have the phenomena of incompleteness, incomplete and even overlapping, and the statistics and the determination of the number, the size and the thickness of the vascular bundles near the epidermis of the highland barley are difficult.
In the exploration of the highland barley stem paraffin section technology, a paraffin section method suitable for the section of the highland barley stem is found by improving and adjusting the dehydration time, the wax immersion time, the selection of the paraffin melting point, the exploration of the section expansion method, the dyeing time and the like of the traditional or recorded method. In addition, in the fixing step, the material is completely immersed in the fixing liquid and then vacuumized, so that tissue deterioration can be avoided. If the sticking tablet is added firstly in the spreading process, the sticking tablet is easy to dilute in a warm water tank at 40 ℃, the effect of sticking tablet can not be achieved, and the tissues are easy to drop off simultaneously in the dewaxing process, so the sticking tablet can be added later to play the effect. The section paraffin section of the highland barley stalks obtained by the flaking method is obtained after adjustment, the section paraffin section is complete and continuous, has no defect, no overlap and distinct layers, the highland barley epidermal cells are smooth and complete, the number of vascular bundles can be clearly and accurately counted and analyzed, the size and the thickness of the vascular bundles can be measured, and the analysis on the anatomical structures of the highland barley stalks is accurate (see fig. 1 and fig. 2).
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (10)
1. A method for slicing highland barley stem profile paraffin suitable for plateau environment is characterized by comprising the six steps of material taking and fixing, dehydration and transparency, waxing and embedding, slicing and spreading, dewaxing and dyeing, mounting and observing.
2. The slicing method according to claim 1, wherein the drawing and fixing method is as follows: and (3) taking a second internode of the base part of the highland barley stalk to be 2-4cm, quickly immersing the highland barley stalk into FAA fixing liquid, vacuumizing and standing the highland barley stalk to obtain a highland barley stalk sample, and fixing the highland barley stalk sample in a refrigerator at 4 ℃ for more than 24 hours.
3. The slicing method according to claim 1, wherein the dehydrating and transparentizing method is as follows: washing fixed highland barley stalks with 50% ethanol for 3 times, 1h each time, treating the washed highland barley internodes with 50% ethanol, 70% ethanol, 80% ethanol and 95% ethanol for 1h respectively, then transferring to absolute ethanol for 1.5h, and finishing dehydration; and (3) sequentially placing the dehydrated highland barley material into absolute ethyl alcohol, mixed solution of xylene and absolute ethyl alcohol which are mixed in equal volume and pure xylene for two times for treatment, so that the material is transparent.
4. The sectioning method according to claim 1, wherein the wax dipping and embedding method is as follows: selecting paraffin with melting point of 58-60 deg.C, dissolving and filtering, transferring transparent highland barley internode into mixed liquid incubator containing xylene and paraffin, and maintaining temperature, adding paraffin for several times; and putting the material after being soaked in the wax into the center of two ends of the embedding box, and slowly putting the embedding box into cold water to solidify the paraffin.
5. The slicing method according to claim 1, wherein the slicing and spreading method is as follows: trimming the embedded material, fixing the embedded material on a slicer, manually turning a slicer pulley, adjusting the slice thickness to 10-20 μm, cutting a complete wax tape, putting the cut wax tape into constant-temperature water at 40 ℃ to completely unfold the wax tape, inserting a glass slide into the lower part of the wax tape in a water tank at 40-45 ℃, slightly taking out the wax tape, uniformly and flatly paving a complete tissue on the glass slide, slowly dripping a sticking piece along the space between the glass slide and the wax tape, and air-drying in a natural environment to obtain a dry slice.
6. The sectioning method according to claim 1, wherein the dewaxing and staining method is as follows: respectively placing the dried slices into dimethylbenzene, mixed solution of dimethylbenzene and absolute ethyl alcohol which are mixed in equal volume, absolute ethyl alcohol, ethyl alcohol with the mass fraction of 95%, ethyl alcohol with the mass fraction of 80%, ethyl alcohol with the mass fraction of 70% and ethyl alcohol with the mass fraction of 50% for treatment for 5min until dewaxing completely; and then placing the dewaxed slices into a 1% safranin aqueous solution for dyeing for 30min, then placing the dyed slices into a 1% fast green solution for dyeing for 5s, and then sequentially placing the slices into 95% ethanol, absolute ethanol, a mixed solution of xylene and absolute ethanol with the same volume and xylene for 3min each time, thus obtaining dyed slices.
7. The method according to claim 2, wherein the FAA fixative is prepared by uniformly mixing 84 parts by volume of 70% alcohol, 5 parts by volume of formaldehyde, 5 parts by volume of glacial acetic acid (glacial acetic acid) and 6 parts by volume of glycerol to obtain the FAA fixative for later use.
8. The method of claim 3, wherein the mixed solution of absolute ethanol and xylene is prepared by mixing absolute ethanol at a volume ratio of 1 and xylene at a volume ratio of 1; and (3) sequentially placing the dehydrated highland barley material into absolute ethyl alcohol, mixed solution of xylene and absolute ethyl alcohol which are mixed in equal volume, pure xylene and pure xylene for treatment, wherein the treatment in each step is carried out for 1-2 hours, so that the material is transparent.
9. The method according to claim 4, wherein the heat preservation is carried out by keeping the temperature of the incubator constant at 58-62 ℃ for 24h, adding pure paraffin once every 6h, and finishing the paraffin addition in 3 times.
10. The method of claim 5, wherein the sticking tablet is prepared by: mixing gelatin 1g, distilled water 100ml, and glycerol 15ml to obtain sticky tablet.
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