CN105973673A - Paraffin sectioning method for eucalyptus tissue - Google Patents

Abstract

The invention belongs to the technical field of plant microscopic observation, and discloses a method for paraffin sectioning eucalyptus tissue. The method includes sampling and fixation, washing, dehydration, transparency, paraffin penetration, embedding, sectioning, spreading, patching and baking, dewaxing and rehydration, dyeing, dehydration and counterstaining, and sealing. The present invention adopts a new type of fixative polyhydroxyacrylic acid, which has a fast fixation speed, can greatly reduce the fixation time, and can make the fixation more uniform; at the same time, we use a completely non-toxic TO transparent agent instead of xylene, and the TO transparent agent is kept at 25°C The above can be miscible with 95% ethanol, can dissolve paraffin and sealing gum, has good volatility, has no adverse effect on the dyeing of common biological dyes, and has good transparent and dewaxing effects. Intact; also use mountant CVmount instead of neutral gum. By optimizing the steps of fixation, dehydration and sealing in paraffin wax slice production, the present invention shortens the slice production period by at least 72 hours on the premise of ensuring the slice effect.

Description

A kind of paraffin section method of eucalyptus tissue

technical field

The invention belongs to the technical field of plant microscopic observation, in particular to a method for paraffin sectioning Eucalyptus (Eucalyptus) tissue.

Background technique

The application of paraffin section technology has a history of more than 300 years, and it is one of the most widely used methods in the routine preparation technology of plant histology. Paraffin section can not only be used to observe the morphological structure of normal plant cells and tissues, but also the main method for observing, researching and judging the morphological changes of cells and tissues in pathology and forensic science, and it has also been widely used in basic biology and other fields. research in the subject area. In recent years, with the development of biological science and technology, and the rise of plant ecological anatomy and molecular biology, paraffin section technology has been more actively used in the field of plant science research, and has become an important tool for the study of ecological adaptability of plants in different habitats or succession series of dominant species. important means.

Paraffin section technology includes the steps of sampling, fixing, washing, dehydration, clearing, paraffin dipping, embedding, slicing, patching, dewaxing, staining, dehydration, clearing, and sealing. The source and location of the material should be selected according to the requirements. The material must be fresh. If it is left for too long, it will cause protein decomposition and denaturation, resulting in cell autolysis and bacterial growth, and cannot reflect the morphology of the tissue in vivo. Fixation refers to immersing the fresh material cut into small pieces with an appropriate fixative solution, rapidly coagulating or precipitating the material components in cells and tissues, terminating all metabolic processes of cells, preventing cell autolysis or tissue changes, and keeping the materials as much as possible when they were taken. Structure. Fixation can harden the tissue, which is conducive to the sectioning, and has the effect of media immersion, which is beneficial to the coloring of the tissue. Washing is to wash away the fixative and its crystal precipitation left in the tissue, otherwise it will affect the later staining effect. Dehydration is the use of certain reagents to remove excess water in the tissue, which is conducive to the immersion of transparent, wax-immersed and embedding reagents. Transparency refers to the use of a transparent agent to replace the dehydrating agent in the tissue, making the material transparent and clean, and increasing the refractive index so that the paraffin can penetrate into the tissue. Wax immersion is to replace the transparent agent with paraffin, so that the paraffin can be immersed in the tissue and play a supporting role, so that the material can be used for sectioning. The purpose of staining is to make different structures in cell tissues present different colors for easy observation. After staining, different organelles, contents and different types of cell tissues in cells can be displayed. The stained sections cannot be observed under a microscope yet, they need to be dehydrated by graded alcohol, transparent with xylene, add an appropriate amount of neutral gum dropwise, and seal the coverslip to make a permanent slide specimen, which can be used for a long time under a light microscope. Observe repeatedly. Generally, it takes 5 to 7 days for a general tissue to be taken, fixed, and sealed to make a slide specimen.

The commonly used fixatives for paraffin sections mainly include FAA fixative (mainly composed of formalin, acetic acid and alcohol, fixed for 8-24 hours), FPA fixative (mainly composed of formalin, propionic acid and alcohol, fixed for 8-24 hours). 24h), Carnoy's fixative (the main components are alcohol and acetic acid; alcohol, acetic acid and chloroform; methanol, acetic acid and chloroform; root tips and anthers are fixed for 15-30 minutes, other materials are fixed for no more than 24 hours), chromium Acid-acetic acid fixative (mainly composed of chromic acid, acetic acid and maltose, fixed for 12-24 hours), Navashin's fixed solution (mainly composed of chromic acid, acetic acid and formaldehyde, fixed for 12-24 hours). Among them, formaldehyde, methanol, ethanol, acetic acid and chloroform are volatile, and formaldehyde, methanol and chloroform are toxic to human body and pollute the environment. The fixation time of conventional fixatives is generally longer, usually between 8h and 24h. During the whole slice making process, steps such as tissue transparency, section dewaxing, and section transparency are all inseparable from xylene, which is a necessary reagent for conventional plant sectioning techniques. However, xylene is a highly volatile and highly toxic chemical reagent, which has a strong toxic effect on human breathing, hematopoiesis and nervous system, and is also one of the carcinogens, seriously endangering the health of those who have been engaged in tissue section experiments for a long time; in addition, two When toluene is used for slicing, it has a strong ability to penetrate tissue, and it is difficult to grasp the clearing time. It is easy to make the sliced tissue shrink, become hard and brittle, increase the difficulty of slicing, and easily form waste pieces and waste materials. Therefore, it is of great significance to find non-toxic substitutes for xylene to improve the working environment of paraffin sections, protect the health of operators and improve the quality of sections.

Contents of the invention

In order to overcome the shortcoming and deficiency of above-mentioned prior art, the primary purpose of the present invention is to provide a kind of paraffin section method of eucalyptus tissue. This method is optimized in the steps of fixation, dehydration, and mounting. Polyhydroxyacrylic acid is used as a fixative, TO transparent agent is used instead of xylene, and mounting agent CVmount is used instead of neutral gum. Time spent in production.

The object of the present invention is achieved through the following solutions:

A kind of paraffin section method of eucalyptus tissue, mainly comprises the following steps:

(1) Material collection and fixation: take 0.3-0.7cm long eucalyptus young tender stem section or primary root as the fixed material, put the material into a glass bottle equipped with polyhydroxyacrylic acid fixative, seal and vacuumize, then statically and fixed at room temperature for 1 h.

(2) Washing: Take out the fixed materials, transfer them into glass bottles filled with 50% ethanol, 70% ethanol, and 70% ethanol in turn, stopper the bottle stoppers and wash them for 13-17 minutes respectively, wherein the liquid volume in the glass bottles Both are more than 30 times that of the material, and then the material is transferred into a glass bottle filled with 70% ethanol for future use. At the same time, 0.05-0.20 mL of 1% safranin alcohol solution is added to identify the material during embedding.

(3) dehydration: the material after washing in step (2) is moved successively into the glass bottles filled with 85% ethanol, 95% ethanol, dehydrated ethanol and dehydrated ethanol whose volume is more than 30 times of the material, and dehydrated for 40-40 ~ 60min.

(4) Transparency: transfer the dehydrated materials in step (3) into glass bottles filled with clearing agent I, clearing agent II, and clearing agent III in turn for 1 hour.

(5) Wax penetration: add broken wax equal to the volume of clearing agent III in the bottle to the glass bottle after the transparent treatment in step (4), then put into an air thermostat, the treatment temperature is 40°C, and the treatment time is 8h; After 8 hours, adjust the temperature of the air incubator to 60°C, and the treatment time is 2 hours; then move the material into pre-heated and melted pure paraffin, and after 4 hours of wax penetration treatment at 60°C, replace the melted pure paraffin, and continue to heat in the air incubator at 60°C Wax penetration treatment for 2h.

(6) Embedding: Pour the melted paraffin into the embedding carton, quickly take out the wax-penetrated material in step (5) and put it into the embedding carton, and adjust the position of the material with a dissecting needle according to the needs of sectioning; After the surface of the paraffin embedded in the carton forms a layer of wax film, the carton is cooled at 2-6°C for 2 hours to make the paraffin solidify from the solution into a paraffin block.

(7) Slicing: the paraffin block embedded in step (6) is cut, trimmed and fixed, then the paraffin block is cut into wax strips through a microtome, the slice thickness is 8-15 μm, and the wax strip is preserved at 30% in ethanol.

(8) Unfolding: Put the wax tape sliced in step (7) into warm water at 30°C to 40°C for 90 to 120 seconds, so that the wax tape is fully unfolded, with the smooth side facing down. Divide the wax ribbon with a razor blade.

(9) Patches and baked slices: On a clean glass slide, apply the egg white glycerin patch, and stick the divided wax tape on the position where the patch is coated on the slide, with the smooth side of the wax tape facing down ; Then add distilled water to the wax tape to ensure that the wax tape is completely soaked, tilt the glass slide to pour off the excess distilled water, put the sliced glass slide into an air incubator, and dry it at 40°C.

(10) Dewaxing and rehydration: Put the sliced glass slides baked in step (9) into an air incubator at a temperature of 60°C to completely melt the paraffin in the slices, and then place the sliced glass slides The glass slides were transferred to clearing agent III and clearing agent II for dewaxing treatment for 25 minutes respectively, then transferred to clearing agent I for dewaxing treatment for 10 minutes, and then transferred to absolute ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and distilled water were rehydrated for 5 minutes, so that the paraffin wax completely faded away, and only the material left in place on the slide was left, that is, the slide with material.

(11) Staining: transfer the material slide obtained in step (10) into 1% safranin alcohol solution, and stain at room temperature for 22-26 hours.

(12) Dehydration and counterstaining:

A, transfer the slides with materials stained in step (11) into 70% ethanol and 80% ethanol in turn for dehydration treatment for 2 min;

B. Transfer the material slide treated by A into 0.63% fast green alcohol solution for counterstaining for 5 seconds;

C. Transfer the slides with material after B counterstaining to 95% ethanol and absolute ethanol for dehydration treatment for 2 minutes respectively;

D. Transfer the glass slide with materials treated by C into absolute ethanol for dehydration for 1min, then transfer to clearing agent Ⅰ and clearing agent Ⅱ for 5 minutes respectively, and finally transfer to clearing agent Ⅲ for 2 minutes. .

(13) Sealing: Take out the glass slide with material treated in step (12) from the transparent agent III, use filter paper to wipe off the excess transparent agent III other than the material, and when the transparent agent III is not volatilized to dryness, remove the material on the slide Add mounting agent dropwise to the material area on the glass slide, take a clean cover glass, and slowly cover it on the glass slide, so that the mounting agent covers the entire material area, and observe after drying at room temperature for 1 hour.

The amount of liquid used in the above steps (1) to (12) can at least immerse the material; the operations without temperature are all carried out at room temperature.

The volume ratio of the materials used in step (1) to the polyhydroxyacrylic acid fixative is 1: (10-30).

The sealing described in the step (1) refers to sealing with a rubber stopper, and applying vaseline at the place where the rubber stopper contacts the bottle mouth; the described vacuuming refers to pumping air 3 to 5 times with a syringe.

In steps (4), (10), and (12), the transparent agent I is an equal-volume mixed solution of TO transparent agent and absolute ethanol, and the transparent agent II and transparent agent III are both TO transparent agents .

The specific operations of cutting, trimming and fixing described in step (7) are as follows: select a suitable platform according to the microtome, divide the embedded paraffin block, and further trim the wax block into a square platform according to the size of the platform , and then use an alcohol lamp to heat the bottom of the wax block to make it melt slightly, then quickly fix it on the table wood, drop a circle of wax liquid at the contact between the wax block and the table wood, and strengthen the wax block after it solidifies.

The preparation method of the egg white glycerin patch described in step (9): mix the egg white and glycerin equal volumes that have been stirred into foam to obtain the egg white glycerin patch. After adding the preservative sodium salicylate, it can Store for more than 3 months.

The mounting medium described in step (13) is CVmount mounting medium.

Mechanism of the present invention is:

(1) The present invention uses a new type of fixative polyhydroxyacrylic acid, which has strong penetration, can greatly accelerate the fixation speed, and can be fixed evenly; it is not volatile at 25-30°C, can be fixed at high temperatures, and also improves its penetration Speed, thus speeding up the fixing speed, greatly reducing the fixing time from 24h to 1h. Because the main components of Carnot's fixative are glacial acetic acid and ethanol, although high temperature fixation can also increase the speed, it will evaporate and its volume ratio will change after evaporation, resulting in poor fixation effect. Polyhydroxyacrylic acid has not been used in plants, so it is difficult for colleagues who do plant sections to find out its efficacy as a fixative in plants.

(2) Use TO transparent agent instead of xylene. TO transparent agent is a bio-film transparent agent with turpentine as the main component. It has the same solubility characteristics as xylene. It can dissolve both alcohol and paraffin. It can completely replace xylene in tissue transparency, section transparency and Used when paraffin sections are dewaxed. Moreover, the reagent is non-toxic to humans, and can make the entire paraffin section operation process a non-toxic operation. In addition, the TO transparent agent has a soft effect during the film-making process, the film-making material is not easy to be hardened, the film-making is easy to succeed, and it is not easy to break when slicing. There is no obvious difference in quality between the produced paraffin sections and the traditional method.

(3) Use the mounting agent CVmount instead of neutral gum to seal the slides, shortening the drying time from 48h to 1h. The neutral gum used in the traditional method contains xylene, so it needs to be dried for a long time to volatilize the xylene during mounting. CVmount is a new type of embedding material, which has good embedding effect, does not contain xylene, and does not need to leave the time required for volatilizing xylene, so it can greatly shorten the mounting time.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

1) the present invention has used novel fixative polyhydroxyacrylic acid, and this fixative fixation speed is fast, can greatly reduce fixation time, and can make fixation more even; It is difficult for volatilization when 25～30 ℃, can be fixed at high temperature, further Improved fixation efficiency.

2) At the same time, completely non-toxic TO transparent agent is used instead of xylene. TO transparent agent can dissolve ethanol, paraffin and sealing gum, has good volatility, and has no adverse effect on the dyeing of commonly used biological dyes. It can be widely used in plants, animals and The slice preparation of human tissue completely avoids the poison to the operator caused by the use of xylene.

3) Using the mounting agent CVmount instead of neutral gum shortens the mounting time from 48h to 1h.

Description of drawings

Fig. 1 is a comparison diagram of microscopic observation of slices (A, B) prepared by the traditional method and slices (C, D) prepared by the method of the present invention.

detailed description

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

The reagents used in this example can be purchased from the market unless otherwise specified; the amount of liquid used in this example can at least immerse the material; the temperature not indicated in this example can be carried out at room temperature.

Example 1:

(1) Take materials and fix

Adopt eucalyptus tissue culture seedling, remove superfluous tissue such as leaf, callus etc. except stem section, get its basal stem section 0.5cm long. Add polyhydroxyacrylic acid fixative (manufactured by Guangzhou Aifa Biomedical Technology Engineering Co., Ltd., with a fixation speed of 1mm/h, first-class medical Device manufacturer registration certificate number: 20090363, stored at a temperature of 10-30°C), put into the stem segment, and put the bottle stopper on. To prevent air from entering the vial, apply petroleum jelly to the top of the rubber stopper and where the rubber stopper meets the mouth of the vial. The vial is placed on the table, insert the rubber stopper with a syringe with a needle (the piston is pushed to the bottom), but do not go deep into the liquid surface of the fixative, and pump air. After pumping to the maximum extent, pull out the syringe to discharge the air in the syringe. This is Vacuum once. After repeating this operation 4 times, let the material rest for 1 hour.

(2) washing

After the material is fixed in the vial for 1 hour, the cork is opened, the material is taken out and placed in a vial filled with 50% ethanol, the volume of which is at least 30 times that of the material, and the cork is tightly closed. After 15 minutes, the washing solution was replaced with 70% ethanol, and then replaced with 70% ethanol for 15 minutes at an interval of 15 minutes, then transferred to 70% ethanol, and 0.1 mL of 1% safranin alcohol solution was added at the same time.

(3) Dehydration

The materials in the vials were sequentially transferred into 85% ethanol for 1 h dehydration treatment, 95% ethanol dehydration treatment for 1 h, and absolute ethanol (twice) for 40 min dehydration treatment respectively.

(4) Transparent

The dehydrated material was successively moved into an equal-volume mixed solution of TO clarifier and absolute ethanol, TO clarifier (TO type biological tablet clarifier, produced by Rosin Factory in Cenxi County, Guangxi; TO clarifier treatment 2 times) The glass bottles were soaked for 1h respectively.

(5) Through the wax

Stack a piece of filter paper into a cylinder with the bottom closed, put it into a vial containing TO clearing agent, pour the crushed wax equal to the volume of TO clearing agent into the filter paper tube, and cover it tightly. Then, put it into an air thermostat, the treatment temperature is 40° C., and the treatment time is 8 hours. After 8 hours, the temperature of the air incubator was adjusted to 60° C., and the treatment time was 2 hours. Then move the material into pre-heated and melted pure paraffin wax at 60°C for 4 hours, replace the melted pure paraffin, and continue the wax penetration treatment at 60°C for 2 hours in an air thermostat.

(6) Embedding

Pour the melted paraffin into the embedding carton, quickly take out the paraffin-permeable material and put it into the embedding carton, and adjust the position of the material with a dissecting needle according to the needs of sectioning. After the surface of the paraffin embedded in the carton forms a layer of wax film, the carton is cooled at 4°C for 2 hours to solidify the paraffin into a paraffin block.

(7) Waxing

Choose the appropriate table wood according to the slicer, divide the wax block containing the material after embedding, trim the wax block into a square table shape according to the size of the table wood, and then heat the bottom of the wax block with an alcohol lamp to make it melt slightly, and then quickly fix it On the table wood, drop a circle of wax liquid at the contact between the wax block and the table wood, and strengthen the wax block after it solidifies.

(8) slice

Put the table wood with the wax block into the wax block of a microtome (KD202A rotary microtome, produced by Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.), make the cut surface of the wax block parallel to the section, and cut the paraffin block into wax blocks. Strips, wax strips can be temporarily stored in 30% ethanol, section thickness 8μm.

(9) Exhibition

After slicing, transfer the wax strip into warm water at 35°C for 100 seconds, with the smooth side of the wax strip facing down. After the wax tape is spread, the wax tape is divided with a blade.

(10) Chips and Baked Sheets

On a clean glass slide, smear the glycerin protein patch (prepared by mixing equal volumes of egg white and glycerin into foam), and stick the divided wax strips on the slide where the patch has been applied , the smooth side of the wax tape is facing down; then, drop distilled water on the wax tape to ensure that the wax tape is completely soaked, tilt the glass slide, pour off the excess distilled water, and place the sliced glass slide in the air for constant temperature Box, 40 ℃ oven dry.

(11) Dewaxing and rehydration

Put the dried glass slides with slices into an air incubator at a temperature of 60°C to completely melt the paraffin, then transfer the slides with slices into TO transparent agent (twice) for dewaxing respectively Treat for 25 minutes; then transfer to TO transparent agent and absolute ethanol equal proportion mixture for dewaxing treatment for 10 min; then transfer to absolute ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol and distilled water They were rehydrated for 5 minutes respectively. The paraffin wax is completely removed, and only the material left in place is left on the slide, that is, the slide with material.

(12) Dyeing

Place the rehydrated slides with materials into 1% safranin alcohol solution and stain at room temperature for 24 hours.

(13) Dehydration and counterstaining

The stained glass slides with material were sequentially transferred to 70% ethanol and 80% ethanol for dehydration treatment for 2 minutes respectively; the dehydrated glass slides with material were transferred to 0.63% fast green (cas code: 2353-45-9, Purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) in alcohol solution for counterstaining for 5 seconds; then transfer the counterstained material slides to 95% ethanol and absolute ethanol for 2 minutes for dehydration respectively; Dehydrate in absolute ethanol for 1 min; then transfer to a mixture of equal volumes of TO clearing agent and absolute ethanol, and TO clearing agent for dehydration for 5 min; finally transfer to TO clearing agent for dehydration for 2 min.

(14) Mounting

Take the glass slide with material out of the TO transparent agent, wipe off the excess TO transparent agent other than the material with filter paper, and drop 1 drop of mounting agent CVmount (purchased Take a clean cover glass from Leica Microsystems (Shanghai) Trading Co., Ltd., and cover it slowly on the glass slide, so that the CVmount covers the entire material area, and then dry it at room temperature for 1 hour for observation.

Comparative Example 1

The rest of the operations are the same as in Example 1, except that (1) fixation, (3) dehydration and (14) sealing are changed, and the specific changes are as follows:

Carnot's fixative was used as the fixative in step (1), and the fixation time was 24 h, and the rest of the operations were the same as in step (1) in Example 1.

Step (3) was revised as follows: the materials in the vial were successively transferred to 75% ethanol for dehydration treatment for 1 hour, 85% ethanol for 1 hour for dehydration treatment, 95% ethanol for 30 minutes for dehydration treatment, 95% ethanol for 30 minutes for dehydration treatment, absolute ethanol (2 times) ) were dehydrated for 1 h respectively.

In step (14), the mounting agent was replaced with neutral gum, and the drying time at room temperature was 48 hours, and the rest of the operations were the same as in step (14) in Example 1.

Comparing the present invention with the traditional method, the method used in the present invention has been optimized in links such as fixation, dehydration, transparency and sealing, which greatly shortens the time used for slice production. The comparison of the two methods in the production time is shown in Table 1 , as can be seen from Table 1, the method used in the present invention shortens at least 72h than the traditional slicing method.

The present invention adopts a new fixative, transparent agent and mounting agent, which not only improves the experimental environment, completely eliminates the poison of the reagents used in the slicing process to people, obviously shortens the time, and ensures the effect of the film production. The material used to make slices is not easy to harden or break, and there is no obvious difference in quality between the produced paraffin slices and slices obtained by traditional methods, as shown in Figure 1, where A and B are microscopic observations of slices prepared by traditional methods Figure, C, D are the microscopic observation figure of the section prepared by the method of the present invention.

Table 1 Traditional method and method of the present invention make tablet time-consuming comparison

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (9)

1. the paraffin section method of an Eucalyptus tissue, it is characterised in that comprise the following steps:

(1) draw materials and fix: taking the Eucalyptus tender stem segments of 0.3～0.7cm length or primary root as fixing Material, puts in the vial equipped with poly-hydroxy acrylic acid fixative by material, seals and evacuation, then Stand, under room temperature, fix 1h；

(2) washing: will fixing after material take out, be moved into successively equipped with 50% ethanol, 70% ethanol, In the vial of 70% ethanol, stopper bottle stopper and wash 13～17min respectively, the wherein liquid bulk in vial Long-pending be material more than 30 times, the most again material is moved into the vial equipped with 70% ethanol preserves standby With, it is simultaneously introduced the sarranine alcoholic solution of 0.05～0.20mL 1%, in order to during embedding, identify material；

(3) dehydration: being moved into successively by the material after washing in step (2) and filling volume is material 30 times Above 85% ethanol, 95% ethanol, dehydrated alcohol, dehydrated alcohol vial in processed respectively 40～60min；

(4) transparent: the material after step (3) is dehydrated is moved into successively equipped with clarifier I, clarifier II, difference transparent processing 1h in the vial of clarifier III；

(5) saturating wax: add and clarifier III in bottle etc. in vial after transparent processing in step (4) The broken wax of volume, is then placed in air incubator, and treatment temperature is 40 DEG C, and the process time is 8h；8h it After the temperature of air incubator is adjusted to 60 DEG C, the process time is 2h；Then material is moved into and add in advance The paraffin refined wax of heat fusing, changes the paraffin refined wax of fusing after 60 DEG C of saturating Lasaxing Oilfield 4h, continue at air incubator In 60 DEG C of saturating Lasaxing Oilfield 2h；

(6) embedding: the paraffin melted is poured into embedding carton, takes out rapidly in step (5) after saturating wax Embedding carton put into by material, according to the position needing to adjust material with dissecting needle cut into slices；In carton to be embedded Paraffin surface form coating of wax film after carton is cooled down 2h under 2～6 DEG C of refrigerated conditions, make paraffin by molten Liquid is frozen into paraffin mass；

(7) section: by the paraffin mass after embedding in step (6) through cutting, finishing and set, then Paraffin mass is cut into wax band by sliced machine, and slice thickness is 8～15 μm, is saved in 30% ethanol by wax band；

(8) exhibition sheet: the wax band after step (7) is cut into slices is put into 30 DEG C～40 DEG C of warm water exhibition sheets 90～120s, Making wax band fully deployed, wax band smooth faces down, and splits wax band with blade after wax band exhibition sheet；

(9) paster and roasting sheet: in clean glass slide, is coated with and wipes Ovum Gallus domesticus album glycerol paster agent, by segmentation well Wax band be attached to microscope slide and scribble on the position of paster agent, wax band smooth faces down；Then drip on wax band Add distilled water, it is ensured that wax band is outwelled unnecessary distilled water by complete dipped rear-inclined microscope slide, will post section Microscope slide put into air incubator, dry for 40 DEG C；

(10) dewaxing rehydration: the microscope slide posting section dried in step (9) is put into air constant temperature In case, treatment temperature is 60 DEG C, makes the paraffin in section be completely melt, then will post the load glass of section Sheet proceeds to difference dewaxing treatment 25min in clarifier III, clarifier II successively, then is transferred in clarifier I Dewaxing treatment 10min, then proceed to successively dehydrated alcohol, 95% ethanol, 90% ethanol, 80% ethanol, 70% In ethanol and distilled water, rehydration processes 5min respectively, makes paraffin take off completely, microscope slide is only left stay Material in situ, i.e. carrying material microscope slide；

(11) dyeing: the carrying material microscope slide obtained in step (10) is proceeded to 1% sarranine alcoholic solution, Dyeing 22～26h under room temperature；

(12) it is dehydrated and redyes:

A, by through step (11) dye after carrying material microscope slide proceed to 70% ethanol, 80% ethanol successively Middle processed 2min respectively；

B, proceed to the carrying material microscope slide processed through A the fast green alcoholic solution of 0.63% redyes 5s；

C, proceed to successively 95% ethanol, dehydrated alcohol take off respectively by the carrying material microscope slide after B redyes Water processes 2min；

D, the carrying material microscope slide processed through C is proceeded to processed 1min in dehydrated alcohol, then depend on Secondary proceed in clarifier I, clarifier II transparent processing 5min respectively, finally proceed in clarifier III transparent Process 2min；

(13) mounting: will take out from clarifier III through the carrying material microscope slide after step (12) processes, Wipe clarifier III unnecessary beyond material with filter paper, clarifier III non-volatile dry time, microscope slide has The region dropping mountant of material, takes the coverslip of cleaning, slowly covers on microscope slide, make mountant be covered with The whole region having material, is observable after dry in the sun 1h under room temperature.

The paraffin section method of Eucalyptus tissue the most according to claim 1, it is characterised in that:

In step (1)～(12), the operation of unreceipted temperature is the most at room temperature carried out.

The paraffin section method of Eucalyptus tissue the most according to claim 1, it is characterised in that:

In step (1)～(12), the amount of liquid used the most at least can submergence material.

The paraffin section method of Eucalyptus tissue the most according to claim 1, it is characterised in that:

Material used in step (1) is 1:(10～30 with the volume ratio of poly-hydroxy acrylic acid fixative).

The paraffin section method of Eucalyptus tissue the most according to claim 1, it is characterised in that:

Sealing described in step (1) refers to seal with rubber closure, and the ground contacted with bottleneck at rubber closure Vaseline is smeared by side；Described evacuation refers to bleed 3～5 times with syringe.

The paraffin section method of Eucalyptus tissue the most according to claim 1, it is characterised in that:

In step (4), (10), (12), described clarifier I is TO clarifier and dehydrated alcohol Equal-volume mixed liquor, described clarifier II and clarifier III are TO clarifier.

The paraffin section method of Eucalyptus tissue the most according to claim 1, it is characterised in that:

The concrete operations of cutting, finishing and the set described in step (7) are as follows: select according to microtome Suitably platform wood, by the paraffin mass segmentation after embedding, is trimmed to four ribs further according to platform wooden rule cun by wax stone Mesa-shaped, then with after making it slightly molten bottom alcohol burner heating wax stone, anchors at rapidly on platform wood, at wax stone and A circle wax liquid is dripped in platform wood contact position, treats that it solidifies post-reinforcing wax stone.

The paraffin section method of Eucalyptus tissue the most according to claim 1, it is characterised in that:

Ovum Gallus domesticus album glycerol paster agent described in step (9) is prepared by following steps: will stir into cystose Ovum Gallus domesticus album and glycerol equal-volume mix and i.e. obtain Ovum Gallus domesticus album glycerol paster agent.

The paraffin section method of Eucalyptus tissue the most according to claim 1, it is characterised in that:

Mountant described in step (13) is CVmount mountant.