CN114088480A

CN114088480A - Preparation method of paraffin sections of root and stem tissues of asafetida

Info

Publication number: CN114088480A
Application number: CN202010861376.7A
Authority: CN
Inventors: 马颖; 周哲之; 朱芸; 刘朦朦; 刘双双; 刘静文; 胡若晰; 王楠; 赵媛媛; 张思维
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2020-08-25
Filing date: 2020-08-25
Publication date: 2022-02-25

Abstract

The invention relates to a preparation method of paraffin sections of root and stem tissues of asafetida, belonging to the technical field of biological histology. The method comprises eleven steps of material taking, fixing, washing, dehydrating, transparentizing, wax dipping, embedding, sheet baking, slicing, sheet baking, dyeing and sheet sealing. Microwave technology is used in the steps of dehydration, transparence and wax dipping, so that the time required for tabletting is greatly shortened. And a safe, environment-friendly and efficient dehydrating and clearing agent capable of replacing dimethylbenzene is used in the clearing step. The method for quickly slicing the asafetida root and stem tissues by paraffin is quick, safe and practical, has strong operability, and is high in repeatability of obtained results, stable and reliable, the quality and the effect of microscopic observation on slicing are high, the tissue structure is clear, and the time is saved on the basis of ensuring the slicing effect.

Description

Preparation method of paraffin sections of root and stem tissues of asafetida

Technical Field

The invention relates to a method for preparing paraffin sections of asafetida root and stem tissues, belongs to the technical field of biological histology, and particularly relates to a method for quickly preparing paraffin sections of asafetida root and stem tissues.

Background

Ferula Ferula structures (Steud.) Korov. is a herb of Ferula genus of Umbelliferae with perennial fruiting, and is widely distributed in the edge region of Quercus angustifolia basin in Sinkiang, and its root and plant resin are used as medicines, and have no scallion and garlic odor, and have effects of resolving food stagnation, killing parasite, eliminating dampness, relieving pain, and treating dental caries.

The paraffin section method is often used for clinical pathological diagnosis and plant anatomical feature observation, is used for observing and judging morphological change of cell tissues, has the advantages of thin section, clear observation of cells, controllable dyeing condition, permanent storage and the like, is the most common method in the conventional histology section making technology, but the traditional paraffin section making period is long, the operation process is complex, more consumables are needed, 3 kinds of carcinogen dimethylbenzene can be generally used, and the nerve system dysfunction can be caused by long-time contact.

The method for preparing the paraffin section by the microwave method is characterized in that under the irradiation of short-wave high-frequency electromagnetic waves, polar molecules and ions vibrate at a high speed and collide with each other to generate a large amount of heat energy, so that tissue cells or media are heated uniformly and uniformly, the liquid exchange efficiency between a reagent and the tissue cells is also improved obviously, and the section preparation time is shortened. In addition, the xylene reagent in the steps of transparence and wax dipping is replaced by the n-butyl alcohol which also has the tissue softening effect, so that not only is the complete tissue section cut out favorably, but also the use of toxic reagents is reduced, in addition, the density of the n-butyl alcohol is smaller than that of molten paraffin, and the n-butyl alcohol is easy to replace by the molten paraffin during embedding, so that the steps of transparence, wax dipping, embedding and the like are simplified. In addition, the method changes the clearing agent into a solution with the mixed concentration gradient of absolute ethyl alcohol and n-butyl alcohol, and then uses the n-butyl alcohol for treatment, so that the water in the tissue can be completely removed, the treatment time of the absolute ethyl alcohol in the dehydration process is shortened, and the problem that the tissue becomes brittle after long-time use of the absolute ethyl alcohol can be avoided.

The root and stem of ferula is used as main test materials, and the main technical links of the traditional plant paraffin slicing method are researched for a long time and improved greatly; the n-butyl alcohol is successfully used for replacing dimethylbenzene as a clearing agent, so that the use of toxic reagents is reduced, the quality of paraffin sections is improved, the use of a microwave method improves the liquid exchange efficiency between the reagents and cell tissues, and the time for manufacturing the paraffin sections is greatly shortened; in conclusion, the microwave rapid paraffin section method has the advantages that research instruments are convenient and easy to obtain, the section making time is greatly shortened, and the method is an efficient and rapid method for researching the root and stem tissues of the asafetida.

Disclosure of Invention

The invention aims to improve the traditional paraffin slicing method, solve the problem of long pre-slicing processing time and shorten the slicing time by adopting a microwave improved paraffin slicing method for roots and stems of ferula.

The technical scheme of the experiment is as follows:

1. material taking and fixing: taking ferula root and stem, placing in FAA stationary liquid (containing formalin-70% ethanol-acetic acid at a ratio of 1: 18: 1), vacuum-fixing, vacuum-pumping with suction pump until there is no bubble in the liquid (about 20 min), and storing overnight.

2. Washing: the embedding box is taken to store the tissue fluid and is washed in running water for 3 d.

3. And (3) dehydrating: adding 150 mL of water and a proper amount of zeolite into a 500 mL beaker, placing the beaker into a microwave oven, heating the beaker in a thawing stage for 10 min (preheating), placing a 25 mL small beaker into the large beaker, pouring 15 mL of dehydrating agent 50% ethanol, and placing the beaker into the microwave oven for heating for 6 min. Then sequentially transferring the plant tissues into 60% ethanol, 70% ethanol, 80% ethanol and 95% ethanol for gradient dehydration, wherein the gradient treatment methods are as above, and each time the dehydrating agent is replaced, the previous dehydrating agent is required to be kept in a small beaker to ensure that the plant is in a wet state. Then adding absolute ethyl alcohol for microwave treatment for 3 min, and finally replacing the absolute ethyl alcohol for microwave heating for 3 min to completely dehydrate.

Note that: and (4) taking out the small beaker after the first dehydration step, observing the amount of the dehydrating agent and the condition of the tissue body, and generating white marks if the dehydrating agent is dehydrated. Note that the amount of water in the beaker is observed and cannot be below 100 mL.

4. And (3) transparency: the n-butanol can be dissolved with water, can be dehydrated again and has transparent effect, and is prepared into a solution with volume ratio of anhydrous ethanol to n-butanol of 3: 1, plant body is placed into 15 mL of the solution and is placed into a small beaker, and the small beaker is placed into a large beaker filled with sufficient water for microwave treatment for 6 min. Sequentially putting plant tissue into solution of anhydrous ethanol and n-butanol at volume ratio of 1: 1 and solution of anhydrous ethanol and n-butanol at volume ratio of 1: 3, and putting into n-butanol for 2 times for transparency.

5. Wax dipping: preparing 15 mL of solution with volume ratio of n-butanol to paraffin of 2: 1, solution with volume ratio of n-butanol to paraffin of 1: 1, and solution with volume ratio of n-butanol to paraffin of 1: 2, and sequentially transferring transparent material into the above solutions. Then, the plant tissues were placed in paraffin for 3 times respectively for microwave treatment. The microwave treatment is carried out for 6 times (12 min each time) by adopting low fire level.

6. Embedding: embedding the waxed tissue body by using an HB-L4 biological tissue embedding machine, and ensuring the integrity of the wax block without cracks as far as possible.

7. Baking the slices: and (3) placing the wax block in an HK-4 biological tissue spreading and baking machine baking oven to be heated for 2-3 min at 35 ℃ until the paraffin surface is slightly hotter than the palm.

8. Slicing: and trimming the paraffin blocks to obtain experimental material wax blocks with flat cut surfaces and rectangular or trapezoidal sections, and placing the wax blocks on a rotary slicing machine for slicing, wherein the slicing thickness is 20-40 mu m.

9. Baking slices: and (3) putting the cut slices into an HK-4 biological tissue spreading and baking machine, heating at 45 ℃ until paraffin is melted and is about to flow downwards, taking out and cooling, and repeating the step for 3-4 times to replace long-time baking.

10. Dyeing: the method comprises the steps of dyeing with 1% safranin and 0.5% fast green, immersing slices in dimethylbenzene for 2 min, immersing in a solution of absolute ethyl alcohol and dimethylbenzene in a volume ratio of 1: 1, absolute ethyl alcohol, a 95% ethanol solution, an 85% ethanol solution and a 70% ethanol solution sequentially for 2 min each time, dyeing with 1% safranin for 4 h, taking out the slices after dyeing, immersing the slices in a 50% ethanol solution, a 70% ethanol solution, an 80% ethanol solution and a 95% ethanol solution sequentially for 2 min each time, washing with 0.5% fast green 5 s and distilled water for 10 s, immersing the slices in the absolute ethyl alcohol solution for 2 min after washing, processing the slices with an immersion solution of dimethylbenzene and absolute ethyl alcohol in a volume ratio of 1: 1 for 5 min, and finally immersing the slices with dimethylbenzene for 5 min.

11. Sealing: the sections were removed from the xylene, 1 drop of neutral canadian gum was dropped into the center of the material before the xylene was completely volatilized, the cover slip side was placed on the tissue section with the gum dropped, the cover slip was slowly lowered and the air was completely removed. The sealed slices are dried at room temperature to form permanent slices.

The invention has the following outstanding advantages

1. The microwave technology is adopted in the steps of dehydrating, transparentizing and waxing the paraffin section, so that the liquid exchange efficiency between the reagent and the cell tissue is improved, the time required by section manufacturing is shortened, the obtained section has high quality and good effect, and the tissue structure is clear. On the premise of ensuring the high quality of the slices, the manufacturing efficiency is greatly improved.

2. The invention provides a method for preparing a tissue clearing agent by using n-butyl alcohol, which is characterized in that a clearing agent is changed into a mixed concentration gradient solution of absolute ethyl alcohol and n-butyl alcohol, and then the mixed concentration gradient solution is treated by the n-butyl alcohol, so that the water in the tissue can be completely removed, the treatment time of the absolute ethyl alcohol in the dehydration process is shortened, the problem that the tissue becomes brittle due to long-time use of the absolute ethyl alcohol can be avoided, the use of a toxic reagent xylene is reduced, and the harm to operators is reduced.

3. In the baking process, the paraffin is heated to 45 ℃ until the paraffin is melted and is taken out for cooling when the paraffin is about to flow downwards, the step is repeated for 3-4 times, the operation of baking the sheet for a long time can be replaced, and the sheet is tightly attached to the glass slide in a short time so as to prevent the sheet from falling.

4. According to the invention, xylene is dropwise added in the embedding process, so that wax blocks can be softened, and the damage of the blade to a wax belt is reduced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a view showing the slicing effect of the paraffin sectioning method of asafetida root tissues according to example 1 of the present invention at 10X 10;

FIG. 2 shows the slicing effect obtained by the paraffin slicing method of asafetida stem tissue according to the present invention in example 2, 10X 10;

FIG. 3 is a graph of 10X 10 showing the effect of paraffin sectioning in a conventional paraffin sectioning operation of comparative example 1;

fig. 4 shows the paraffin sectioning effect obtained in the conventional paraffin sectioning operation of comparative example 2, 10 × 10.

Detailed Description

The raw materials and reagents used in the paraffin sectioning method of plant tissues provided by the invention can be purchased from the market.

The invention is further illustrated below with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Example one

1. Material taking and fixing: cutting the root of resina Ferulae into small segments with a length of about 3 mm, placing into a glass bottle, adding FAA fixative (containing formalin-70% ethanol-acetic acid at a ratio of 1: 18: 1) to kill cells with reagent, fixing shape, and determining that the fixative covers tissue without exceeding 4/5 of the volume of the glass bottle to prevent incomplete fixation of tissue due to extraction of the fixative. Vacuuming for about 20 min (no air bubbles generated) and storing overnight.

2. Washing: the embedding box is taken to store the tissue fluid and washed in flowing water for 3 d.

3. And (3) dehydrating: and (3) dehydrating: soaking and dehydrating with gradient ethanol at room temperature. The ethanol gradient is 70% ethanol solution, 80% ethanol solution, 95% ethanol solution, anhydrous ethanol, and anhydrous ethanol in sequence. Adding 150 mL of water and a proper amount of zeolite into a 500 mL big beaker, then placing the beaker into a microwave oven, heating the beaker in a thawing mode for 10 min (preheating), placing a 25 mL small beaker into the big beaker, pouring 15 mL of dehydrating agent 70% ethanol, heating the beaker in a heat preservation mode for 6 min, taking out the beaker, observing the amount of the dehydrating agent and the condition of tissue bodies, and if white marks occur in dehydration, observing the amount of the dehydrating agent and the water amount which cannot be lower than 100 mL. And (3) sequentially transferring the plant tissues into an ethanol solution with the concentration of 80% and an ethanol solution with the concentration of 95% to perform gradient dehydration, wherein a little dehydrating agent is remained in a small beaker in the previous time to ensure that the plant is in a wet state when the dehydrating agent is replaced each time. After the 85% ethanol solution is added, violent liquid exchange is generated, and the plants bubble, which indicates that the cavitation effect exists. Finally, absolute ethyl alcohol is changed for 2 times, and the heating is carried out for 3 min each time. The detection method comprises the following steps: the plant body is put into a dimethylbenzene solution, whether water mist occurs or not is observed, and if the water mist occurs, dehydration is not thorough.

4. And (3) transparency: the clearing agent is mixed solution of n-butanol and anhydrous ethanol and n-butanol, and the solution used in the clearing process is sequentially solution of anhydrous ethanol and n-butanol at a volume ratio of 3: 1, solution of anhydrous ethanol and n-butanol at a volume ratio of 1: 3, n-butanol solution, and n-butanol solution. The n-butyl alcohol can be mutually soluble with water, not only can be dehydrated again, but also can play a role of transparency, and 15 mL of the reagents are replaced in sequence in a small beaker and placed in a large beaker filled with 150 mL of water. The root segment of resina Ferulae is placed in a small beaker, and the whole is placed in a microwave oven together with a large beaker for heating for 6 min for transparency.

5. Wax dipping: preparing a solution with a volume ratio of n-butanol to paraffin of 2: 1, a solution with a volume ratio of n-butanol to paraffin of 1: 2 and two parts of molten paraffin each 15 mL in penicillin bottles, sequentially transferring the transparent materials into the corresponding penicillin bottles, and placing the penicillin bottles in a large beaker containing water for low-fire microwave treatment for 12 min each time. And the water level and the water supplement are observed in time.

6. Embedding: after paraffin in an embedding machine is melted, molten paraffin is dripped until the bottom of an embedding box is completely covered, a drop of dimethylbenzene is dripped to be uniformly stirred, a part of paraffin is added, plant bodies are added (the plant bodies are vertically placed to keep distance among the plant bodies), when the paraffin is in a semi-solidification state, the paraffin is continuously dripped until the paraffin in the embedding box is full, the embedding box is covered, and the paraffin is dripped again until the paraffin is full. Finally, the embedding box is moved to a 4 ℃ freezing table to be rapidly cooled and solidified, and finally the wax block is dismounted.

7. Baking the slices: and (3) placing the wax block in an HK-4 biological tissue spreading and baking machine baking oven for heating at 35 ℃ for 2-3 min until the paraffin surface is slightly hotter than the palm, or neutralizing in hot water.

8. Slicing: and placing the wax blocks on a rotary slicing machine for slicing, wherein the slicing thickness is 20-40 mu m.

11. Sealing: the sections were removed from the xylene solution, 1 drop of neutral canadian gum was dropped into the center of the material before the xylene was completely volatilized, the cover slip side was placed on the tissue section with the gum dropped, the cover slip was slowly lowered and the air was completely removed. The sealed slices are dried at room temperature to form permanent slices.

Example two

1. Material taking and fixing: cutting the stem of resina Ferulae into small segments with a length of about 3 mm, placing into a glass bottle, adding FAA fixative (containing formalin-70% ethanol-acetic acid at a ratio of 1: 18: 1) to kill cells with reagent, fixing shape, and determining that the fixative covers the tissue body and does not exceed 4/5 of the volume of the glass bottle to prevent incomplete fixation of the tissue body due to extraction of the fixative. Vacuuming for about 20 min (no air bubbles generated) and storing overnight.

2. As described in example one.

3. And (3) dehydrating: soaking and dehydrating with gradient ethanol at room temperature. The ethanol gradient is 50% ethanol solution, 60% ethanol solution, 70% ethanol solution, 80% ethanol solution, 95% ethanol solution, anhydrous ethanol, and anhydrous ethanol in sequence. Adding 150 mL of water and a proper amount of zeolite into a 500 mL big beaker, then placing the beaker into a microwave oven, heating the beaker in a thawing mode for 10 min (preheating), placing a 25 mL small beaker into the big beaker, pouring 15 mL of dehydrating agent 50% ethanol, heating the beaker in a heat preservation mode for 6 min, taking out the beaker, observing the amount of the dehydrating agent and the condition of tissue bodies, and observing the amount of water if white marks occur in dehydration and keeping the amount of water to be not less than 100 mL. And (3) sequentially transferring the plant tissues into an ethanol solution with the concentration of 60%, an ethanol solution with the concentration of 70%, an ethanol solution with the concentration of 80% and an ethanol solution with the concentration of 95% to perform gradient dehydration, wherein a small amount of the previous dehydrating agent is kept in a small beaker to ensure that the plant is in a wet state when the dehydrating agent is replaced each time. After the 80% ethanol solution is added, violent liquid exchange is generated, and the plants bubble, which indicates that the cavitation effect exists. Finally, absolute ethyl alcohol is changed for 2 times, and the heating is carried out for 3 min each time. The detection method comprises the following steps: the plant body is put into a dimethylbenzene solution, whether water mist occurs or not is observed, and if the water mist occurs, dehydration is not thorough.

4. And (3) transparency: the clearing agent is mixed solution of n-butanol and anhydrous ethanol and n-butanol, and the solution used in the clearing process is sequentially solution of anhydrous ethanol and n-butanol at a volume ratio of 3: 1, solution of anhydrous ethanol and n-butanol at a volume ratio of 1: 3, n-butanol solution, and n-butanol solution. The n-butyl alcohol can be mutually soluble with water, not only can be dehydrated again, but also can play a role of transparency, and 15 mL of the reagents are replaced in sequence in a small beaker and placed in a large beaker filled with 150 mL of water. The stem of Ferula ferula is placed in a small beaker, and the whole is placed in a microwave oven together with a large beaker for heating for 6 min for transparence.

5-11 are as described in example one.

Claims

1. A preparation method of paraffin sections of root and stem tissues of asafetida is characterized by comprising the following steps: the method comprises the following steps:

step 1, sample fixation: taking roots and stems of the small sections of the ferula asafoetida, putting the small sections of the ferula asafoetida in a glass bottle, adding the stationary liquid, vacuumizing, fixing and preserving;

step 2, sample washing: taking an embedding box to store the tissue fluid, and flushing the tissue fluid in running water;

step 3, sample dehydration: step-by-step dehydrating the ferula asafetida tissue sample washed in the step 2 by using ethanol with different concentrations, wherein the dehydration concentration gradient is 50% → 60% → 70% → 80% → 95% → 100% → 100%, each step is processed in a microwave oven, and the dehydration time is 3-6 min;

and 4, transparent treatment: performing transparent treatment on the ferula tissue sample dehydrated in the step 3 by using a mixed solution of n-butyl alcohol and absolute ethyl alcohol and n-butyl alcohol;

step 5, wax dipping treatment: performing waxing treatment on the ferula asafoetida tissue sample after being transparent in the step 4 by using a mixed solution of paraffin and n-butyl alcohol and paraffin, wherein the concentration gradient of the waxing treatment is that the volume ratio of the n-butyl alcohol to the paraffin is 2: 1 → the volume ratio of the n-butyl alcohol to the paraffin is 1: 1 → the solution of the n-butyl alcohol to the paraffin is 1: 2 → the paraffin, and the treatment time is 12 min in each stage of treatment in a microwave oven;

step 6, embedding the sample: embedding the resina ferulae tissue sample subjected to wax dipping in the step 5 by using an HB-L4 biological tissue embedding machine, and ensuring that a wax block is complete and has no crack as much as possible;

step 7, baking the slices: baking the wax block obtained after embedding in the step 6 by using an HK-4 biological tissue baking machine, and heating in a baking oven at 35 ℃ for 2-3 min until the paraffin surface is slightly hotter than the palm;

step 8, slicing: trimming a paraffin block to obtain an experimental material wax block with a flat section and a rectangular or trapezoidal section, and placing the wax block on a rotary slicing machine for slicing, wherein the slicing thickness is 20-40 mu m;

step 9, baking slices: putting the ferula asafoetida tissue sample slices obtained in the step 8 into an HK-4 biological tissue roasting machine, heating at 45 ℃ until paraffin is melted and is about to flow downwards, taking out and cooling, repeating the step for 3-4 times, and replacing long-time roasting;

step 10, tissue staining: dewaxing the tissue sample section of the ferula in the step 9, and then dyeing the tissue sample section by using 1% of safranin and 0.5% of fast green;

step 11, sealing: and (3) before xylene in the ferula asafoetida tissue sample slices obtained in the step 10 is completely volatilized, 1 drop of neutral Canadian gum is dripped in the center of the material for sealing.

2. The method of preparing paraffin sections of asafetida root and stem tissues as claimed in claim 1, wherein: in the step 1, the roots and stems of ferula asafetida are cut into small segments with the length of about 3 mm respectively.

3. The method for preparing paraffin sections of asafetida root and stem tissues as claimed in claims 1 to 2, wherein: in step 1, a glass vial containing the fixation fluid and the tissue sample was evacuated with a suction pump until there were no air bubbles in the fluid (about 20 min), and stored overnight.

4. The method for preparing paraffin sections of asafetida root and stem tissues as claimed in claims 1 to 3, wherein: in the step 2, the embedding box for storing the tissue body is washed in running water at the temperature of 20-25 ℃ for 3 days.

5. The method for preparing paraffin sections of the root and stem tissue of Ferula asafoetida as claimed in claims 1 to 4, wherein: in the step 3, the water bath device is preheated for 10 min by using a microwave oven unfreezing gear before dehydration.

6. The method for preparing paraffin sections of the root and stem tissue of Ferula asafoetida as claimed in claims 1 to 5, wherein: and 3, in the step-by-step dehydration process, carrying out microwave heating on the beaker containing the transparent reagent and the sample by using a low-fire gear of a microwave oven.

7. The method of preparing paraffin sections of asafetida root and stem tissues as claimed in claims 1 to 6, wherein: in the step 4, the concentration gradient of the treatment solution in the stepwise transparency process is sequentially anhydrous ethanol and n-butanol at a volume ratio of 3: 1 → anhydrous ethanol and n-butanol at a volume ratio of 1: 3 → n-butanol.

8. The method of preparing paraffin sections of asafetida root and stem tissues as claimed in claim 7, wherein: in the step 4, in the step-by-step transparent process, each step is processed in a microwave oven, and the beaker containing the transparent reagent and the sample is placed in the microwave oven for microwave heating for 6 min.

9. The method for preparing paraffin sections of asafetida root and stem tissues as claimed in claims 1 to 8, wherein: in the step 5, the concentration gradient of the tissue sample treatment solution is sequentially that solution with the volume ratio of n-butanol to paraffin being 2: 1 → solution with the volume ratio of n-butanol to paraffin being 1: 2 → paraffin.

10. The method for preparing paraffin sections of the root and stem tissue of asafetida as claimed in claim 9, wherein: in the step 5, each stage of solution treatment is carried out in a microwave oven by adopting low-fire microwave treatment, and the treatment time is 12 min.

11. The method of preparing paraffin sections of asafetida root and stem tissues as claimed in claims 1 to 10, wherein: and 6, melting the paraffin in the embedding machine, pouring the molten paraffin into an embedding mold, taking the material out of the paraffin dipping box, placing the material in a paraffin liquid in the center of the embedding mold, moving the embedding mold to a 4 ℃ freezing table to be rapidly cooled and solidified, and finally unloading the wax block.

12. The method of claim 11, wherein the paraffin section of the root and stem tissue of asafetida is prepared by the following steps: in the step 6, during pouring the molten paraffin into the embedding mold, a drop of xylene is dropped into the paraffin and stirred uniformly.

13. The method for preparing paraffin sections of asafetida root and stem tissues as claimed in claims 1 to 12, wherein: in the step 9, the paraffin is heated to 45 ℃ until the paraffin is melted and is taken out for cooling when the paraffin is about to flow downwards, and the step is repeated for 3-4 times instead of baking the slices for a long time.

14. The method for preparing paraffin sections of asafetida root and stem tissues as claimed in claims 1 to 13, wherein: in the step 10, the ferula asafetida tissue sample section obtained in the step 9 is stained with 1% safranin and 0.5% fast green, the method comprises the steps of immersing in a solution (2 min) → absolute ethyl alcohol to xylene volume ratio of 1: 1 (2 min) → absolute ethyl alcohol (2 min) → 95% ethyl alcohol solution (2 min) → 85% ethyl alcohol solution (2 min) → 70% ethyl alcohol solution (2 min) → 1% safranine dyeing (4 h) → 50% ethyl alcohol solution (2 min) → 70% ethyl alcohol solution (2 min) → 80% ethyl alcohol solution (2 min) → 95% ethyl alcohol solution (2 min) → 0.5% fast green dyeing (5 s) → distilled water washing (10 s) → absolute ethyl alcohol solution (2 min) → xylene to anhydrous ethyl alcohol solution (5 min) → xylene (5 min) volume ratio of 1: 1).

15. The method of preparing paraffin sections of asafetida root and stem tissues as claimed in claim 14, wherein: in the step 10, dewaxing treatment is required in the dyeing process.

16. The method of preparing paraffin sections of asafetida root and stem tissues as claimed in claims 1 to 15, wherein: in the step 11, after the gum is dripped, one side of the cover glass is placed on the tissue slice dripped with the gum, the cover glass is slowly put down, air is completely removed, and the sealed slice is dried at room temperature to prepare a permanent slice.