Manufacturing method and application of microwave efficient paraffin section of animal tissue

Abstract

The invention discloses a microwave efficient paraffin section manufacturing method for animal tissues and application of the microwave efficient paraffin section manufacturing method. The microwave efficient paraffin section manufacturing method comprises the following steps of sampling, fixing, dewatering, transparentizing, paraffin soaking, embedding, slicing, section unfolding, dyeing, section sealing and observing. According to the method disclosed by the invention, by combining a microwave technology and aiming at the characteristics of zebra fish intestinal tissue materials, the steps of fixing, dehydrating, transparentizing, waxing and the like in the operation process are optimized and improved, so that the experiment time is greatly shortened; taking zebra fish intestinal tissues as an example, the traditional paraffin section preparation needs about 100 hours, but the paraffin section preparation method disclosed by the invention only needs 6 hours, so that the rapid paraffin section preparation is realized, the experiment time is greatly shortened, and the intestinal tissue section with a complete structure can also be obtained.

Description

Technical Field

[0001] The invention relates to the technical field of animal tissue slice preparation, in particular to a method for preparing microwave high-efficiency paraffin slices of animal tissue and application thereof. Background Art

[0002] Tissue sectioning technology is a technology used in tissue embryology and physiological pathology to observe the changes of tissue cells in real time. This technology is the most effective means to reflect the real-time status of tissues. The slices that are closest to the in vivo status can most truly reflect the changes of tissues. Among tissue sections, paraffin sections are the most commonly used. Paraffin sectioning technology (paraffin sectioning) is to use paraffin as an embedding agent, fix, dehydrate, make transparent, and dip the sample in wax, then embed it in paraffin, and then slice it with a slicer, and then go through dewaxing, staining and other steps to make the sample into a permanent glass slide specimen. Paraffin sectioning technology is an important research method for studying histology and developmental biology, and is widely used in morphological observation and pathological diagnosis research of animal and plant samples. The specific production process of conventional paraffin sections is: sampling → fixation → dehydration → transparent → dip in wax → embedding → sectioning → slide spreading → staining → sealing → observation. Each of these steps is interrelated. Any problem in any step will affect the quality of the final preparation and the observation of the results.

[0003] The paraffin section method has many advantages: it is easy to operate, the material can be cut into uniform and continuous slices, and the material can be permanently preserved after being made into paraffin sections. However, due to the characteristics of paraffin materials, the traditional paraffin section method has its disadvantages, such as: the production cycle of this technology is long, the operation process is relatively complicated and cumbersome, and it is easily affected by many factors, making it difficult to produce good sections. Therefore, shortening the production cycle is the direction of improving paraffin section technology.

[0004] Microwaves are a kind of high-frequency electromagnetic waves, not ionizing radiation. They will not break the weakest hydrogen bonds, but they can make the disordered polar molecules and ions vibrate, rub against each other, and collide under the action of microwaves, generating heat, which increases the temperature of tissue cells or media, resulting in a significant increase in the penetration and exchange efficiency between reagent liquid and tissue cells. At present, there are some studies on the application of microwave method to plant experimental materials. For example, Wei Xinyue et al. (2011) used microwave rapid paraffin sectioning method to prepare rice leaf tissue sections, which compressed the time of fixation, dehydration and wax immersion process to 3 hours; Li Zuren et al. (2012) used American pokeweed as experimental material and applied microwave technology to obtain tissue sections with the same effect as traditional paraffin sectioning method. However, so far, there are not many studies on the application of microwave method to animal tissue materials.

[0005] The preparation of animal tissue samples is much more complicated than that of plant samples. This is because animal tissue samples are mostly soft and water-rich tissues, such as the tissues of small animals such as mice. These tissues are more tender and soft than human tissues, and contain more water and less fiber. These characteristics make animal tissues more important to be fixed, dehydrated, transparent, and wax-impregnated. In order to better fix the material and make the material completely dehydrated and wax-impregnated, while shortening the sample processing time and maintaining the integrity of the sample, it is necessary to apply microwave technology to the preparation of animal tissue sections. Zebrafish (Danio rerio) belongs to the teleost fish, Cyprinidae, Cyprinidae, and has the advantages of being easy to raise, small in size, short in sexual maturity, and fast in reproduction. It is one of the five fish experimental animals recognized by the International Organization for Standardization. At the same time, it is also an important vertebrate model with many applications in drug screening, genetic development, environmental toxicology, and neuroscience. In the study of zebrafish, tissue sectioning technology is the most widely used technical means.

[0006] At present, the research on the normal structure of zebrafish at home and abroad mainly focuses on the telencephalon, midbrain and intestines. Through analysis, it is found that researchers have a great demand for zebrafish tissue paraffin sectioning methods. Through literature search, it is found that there is a lack of literature on zebrafish paraffin sections, and the relevant experimental methods are also uneven. Therefore, it is of great significance to develop a microwave efficient paraffin sectioning method for animal tissues and apply this method to zebrafish sections. Summary of the invention

[0007] The purpose of the present invention is to provide a microwave efficient paraffin section preparation method and application of animal tissue in view of the problem of long preparation cycle in animal tissue paraffin section preparation technology.

[0008] The present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for preparing microwave efficient paraffin sections of animal tissues, comprising the following steps:

[0010] (1) Dissection: remove the required animal tissues and sterilize them for later use;

[0011] (2) Fixation: Place the tissue sample in a glass vial and pour in a neutral formaldehyde fixative until the tissue sample is covered; take a 200 mL beaker, add 50 mL of water, and then heat the water to 50°C. Place the glass vial containing the tissue sample in the beaker, put the beaker in a microwave oven, adjust the microwave oven to low heat for 20 minutes, heat for 5 minutes, maintain the water temperature at 50°C, and take out the beaker; replace the fixative and repeat the microwave heating step once; then place the glass vial containing the tissue sample at room temperature for later use;

[0012] (3) Dehydration treatment:

[0013] a) Aspirate the neutral formaldehyde fixative in the glass vial, add 50% v / v ethanol solution until the tissue sample is covered; put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0014] b) aspirate the 50% v / v ethanol solution in the glass vial, add 70% v / v ethanol solution until the tissue sample is covered; place the glass vial in a beaker, place the beaker in a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0015] c) aspirate the 70% v / v ethanol solution in the glass vial, add 85% v / v ethanol solution until the tissue sample is covered; place the glass vial in a beaker, place the beaker in a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0016] d) Aspirate the 85% v / v ethanol solution in the glass vial, add 90% v / v ethanol solution until the tissue sample is covered; put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0017] e) aspirating the 90% v / v ethanol solution in the glass vial, adding anhydrous ethanol solution until the tissue sample is covered; placing the glass vial in a beaker, placing the beaker in a microwave oven, adjusting the microwave oven to low heat setting 20, heating for 5 minutes, maintaining the water temperature at 50°C, taking out the beaker, and letting it stand for 1 minute for subsequent treatment;

[0018] (4) Transparent processing:

[0019] a) Aspirate the anhydrous ethanol solution in the glass vial, add an anhydrous ethanol-xylene mixture with a volume ratio of 1:1 until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0020] b) Aspirate the anhydrous ethanol-xylene mixture in the glass vial, add xylene solution until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0021] c) aspirating the xylene solution in the glass vial and adding new xylene solution until the tissue sample is covered; sealing the glass vial with a sealing film, placing the glass vial in a beaker, placing the beaker in a microwave oven, adjusting the microwave oven to low heat 20, heating for 5 minutes, maintaining the water temperature at 50°C, taking out the beaker, and letting it stand for 1 minute for subsequent treatment;

[0022] (5) Wax treatment

[0023] a) Aspirate the xylene solution in the glass vial, add a mixture of melted paraffin and xylene preheated to 55°C in a volume ratio of 1:1 until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 10 minutes, maintain the water temperature at 55°C, take out the beaker, and let it stand for 1 minute;

[0024] b) aspirating the paraffin-xylene mixture in the glass vial, adding a pre-heated melted paraffin and xylene mixture in a volume ratio of 4:1 until the tissue sample is covered; sealing the glass vial with a sealing film, placing the glass vial in a beaker, placing the beaker in a microwave oven, adjusting the microwave oven to low heat 20, heating for 10 minutes, maintaining the water temperature at 55°C, taking out the beaker, and letting it stand for 1 minute;

[0025] c) aspirate the paraffin-xylene mixture in the glass vial, add the pre-heated melted pure paraffin solution until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 10 minutes, maintain the water temperature at 55°C, take out the beaker, and let it stand for 1 minute;

[0026] d) The pure paraffin solution in the glass vial is sucked out, and new pure paraffin solution that has been pre-heated and melted is added until the tissue sample is covered; after sealing the glass vial with a sealing film, the glass vial is placed in a beaker, and the beaker is placed in a microwave oven, and the microwave oven is adjusted to a low heat setting of 20, heated for 10 minutes, and the water temperature is maintained at 55°C, and the beaker is taken out and allowed to stand for 1 minute for subsequent treatment;

[0027] (6) Embedding treatment

[0028] Pour the tissue material in the glass vial treated above into a paper box of liquid paraffin to cool;

[0029] (7) Sectioning and Spreading

[0030] The solidified paraffin and materials were sliced ​​with a microtome, with a slice thickness of 10 μm, and then the wax strips were placed at 37°C for expansion;

[0031] (8) Staining and sealing

[0032] Use 1% toluidine blue dye solution for staining, specifically: place the slide in the following solutions: xylene solution for 30 minutes → 1:1 volume ratio of anhydrous ethanol-xylene mixture for 90 seconds → anhydrous ethanol solution for 90 seconds → 95% volume fraction ethanol solution for 90 seconds → 85% volume fraction ethanol solution for 90 seconds → 70% volume fraction ethanol solution for 90 seconds → 50% volume fraction ethanol solution for 90 seconds → 30% volume fraction ethanol solution for 90 seconds → distilled water for 90 seconds → Dyeing with 1% toluidine blue dye solution for 90s → distilled water for 90s → 30% ethanol solution for 90s → 50% ethanol solution for 90s → 70% ethanol solution for 90s → 85% ethanol solution for 90s → 95% ethanol solution for 90s → anhydrous ethanol solution for 90s → a 1:1 anhydrous ethanol-xylene mixture for 90s → pure xylene solution, and finally seal the slide with neutral gum and place in a ventilated place to dry.

[0033] The present invention also provides an application of a microwave efficient paraffin sectioning method for animal tissues, wherein the animal tissues are zebrafish intestines, mouse intestines or rabbit intestines.

[0034] The present invention utilizes microwaves that will not break the weakest hydrogen bonds, but can make the polar molecules and ions in disordered arrangement vibrate, rub against each other, and collide under the action of microwaves, thereby generating heat, raising the temperature of tissue cells or medium, and significantly improving the penetration and exchange efficiency between reagent liquid and tissue cells. Therefore, on the basis of the traditional paraffin section method, combined with microwave technology, the steps of fixation, dehydration, transparency and wax dipping in the operation process are optimized and improved according to the characteristics of zebrafish intestinal tissue materials, greatly shortening the experimental time. Taking zebrafish intestinal tissue as an example, the traditional paraffin section preparation requires about 100 hours, while the paraffin section preparation method of the present invention only requires 6 hours, greatly shortening the experimental time, and also obtaining intestinal tissue sections with complete structures.

[0035] Instruction Manual

[0036] Figure 1 The paraffin sections of zebrafish intestinal tissue obtained by microwave paraffin sectioning in Example 1, wherein Figure B is an enlarged view of the boxed area in Figure A;

[0037] Figure 2 These are paraffin sections of zebrafish intestinal tissue obtained using conventional paraffin sectioning in Example 2, wherein Figure D is an enlarged view of the boxed area in Figure C. DETAILED DESCRIPTION

[0038] In order to better explain the present invention and facilitate understanding of the technical solution of the present invention, it is now further described in conjunction with the drawings and embodiments. It should be understood that the specific embodiments of the present invention are only for illustrative purposes and are not intended to limit the present invention.

[0039] Example 1

[0040] The present embodiment provides a microwave efficient paraffin section preparation method for animal tissue, taking zebrafish intestinal tissue as an example, comprising the following steps:

[0041] (1) Dissection: Prepare forceps, scissors, and a scalpel, and disinfect the zebrafish with anhydrous ethanol. Perform dissection on a sterile bench and remove the zebrafish intestinal tissue.

[0042] (2) Fixation: Place the tissue sample in a glass vial and pour in a neutral formaldehyde fixative until the tissue sample is covered; take a 200 mL beaker, add 50 mL of water, and then heat the water to 50°C. Place the glass vial containing the tissue sample in the beaker, put the beaker in a microwave oven, adjust the microwave oven to low heat for 20 minutes, heat for 5 minutes, maintain the water temperature at 50°C, and take out the beaker; replace the fixative and repeat the microwave heating step once; then place the glass vial containing the tissue sample at room temperature for later use;

[0043] (3) Dehydration treatment:

[0044] a) Aspirate the neutral formaldehyde fixative in the glass vial, add 50% v / v ethanol solution until the tissue sample is covered; put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0045] b) aspirate the 50% v / v ethanol solution in the glass vial, add 70% v / v ethanol solution until the tissue sample is covered; place the glass vial in a beaker, place the beaker in a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0046] c) aspirate the 70% v / v ethanol solution in the glass vial, add 85% v / v ethanol solution until the tissue sample is covered; place the glass vial in a beaker, place the beaker in a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0047] d) Aspirate the 85% v / v ethanol solution in the glass vial, add 90% v / v ethanol solution until the tissue sample is covered; put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0048] e) aspirating the 90% v / v ethanol solution in the glass vial, adding anhydrous ethanol solution until the tissue sample is covered; placing the glass vial in a beaker, placing the beaker in a microwave oven, adjusting the microwave oven to low heat setting 20, heating for 5 minutes, maintaining the water temperature at 50°C, taking out the beaker, and letting it stand for 1 minute for subsequent treatment;

[0049] (4) Transparent processing:

[0050] a) Aspirate the anhydrous ethanol solution in the glass vial, add an anhydrous ethanol-xylene mixture with a volume ratio of 1:1 until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0051] b) Aspirate the anhydrous ethanol-xylene mixture in the glass vial, add xylene solution until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 5 minutes, maintain the water temperature at 50°C, take out the beaker, and let it stand for 1 minute;

[0052] c) aspirating the xylene solution in the glass vial and adding new xylene solution until the tissue sample is covered; sealing the glass vial with a sealing film, placing the glass vial in a beaker, placing the beaker in a microwave oven, adjusting the microwave oven to low heat 20, heating for 5 minutes, maintaining the water temperature at 50°C, taking out the beaker, and letting it stand for 1 minute for subsequent treatment;

[0053] (5) Wax treatment

[0054] a) Aspirate the xylene solution in the glass vial, add a mixture of melted paraffin and xylene preheated to 55°C in a volume ratio of 1:1 until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 10 minutes, maintain the water temperature at 55°C, take out the beaker, and let it stand for 1 minute;

[0055] b) aspirating the paraffin-xylene mixture in the glass vial, adding a pre-heated melted paraffin and xylene mixture in a volume ratio of 4:1 until the tissue sample is covered; sealing the glass vial with a sealing film, placing the glass vial in a beaker, placing the beaker in a microwave oven, adjusting the microwave oven to low heat 20, heating for 10 minutes, maintaining the water temperature at 55°C, taking out the beaker, and letting it stand for 1 minute;

[0056] c) aspirate the paraffin-xylene mixture in the glass vial, add the pre-heated melted pure paraffin solution until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, adjust the microwave oven to low heat 20, heat for 10 minutes, maintain the water temperature at 55°C, take out the beaker, and let it stand for 1 minute;

[0057] d) The pure paraffin solution in the glass vial is sucked out, and new pure paraffin solution that has been pre-heated and melted is added until the tissue sample is covered; after sealing the glass vial with a sealing film, the glass vial is placed in a beaker, and the beaker is placed in a microwave oven, and the microwave oven is adjusted to a low heat setting of 20, heated for 10 minutes, and the water temperature is maintained at 55°C, and the beaker is taken out and allowed to stand for 1 minute for subsequent treatment;

[0058] (6) Embedding treatment

[0059] Pour the tissue material in the glass vial treated above into a paper box of liquid paraffin to cool;

[0060] (7) Sectioning and Spreading

[0061] The solidified paraffin and materials were sliced ​​with a microtome, with a slice thickness of 10 μm, and then the wax strips were placed at 37°C for expansion;

[0062] (8) Staining and sealing

[0063] Use 1% toluidine blue dye solution for staining, specifically: place the slide in the following solutions: xylene solution for 30 minutes → 1:1 volume ratio of anhydrous ethanol-xylene mixture for 90 seconds → anhydrous ethanol solution for 90 seconds → 95% volume fraction ethanol solution for 90 seconds → 85% volume fraction ethanol solution for 90 seconds → 70% volume fraction ethanol solution for 90 seconds → 50% volume fraction ethanol solution for 90 seconds → 30% volume fraction ethanol solution for 90 seconds → distilled water for 90 seconds → Dyeing with 1% toluidine blue dye solution for 90s → distilled water for 90s → 30% ethanol solution for 90s → 50% ethanol solution for 90s → 70% ethanol solution for 90s → 85% ethanol solution for 90s → 95% ethanol solution for 90s → anhydrous ethanol solution for 90s → a 1:1 anhydrous ethanol-xylene mixture for 90s → pure xylene solution, and finally seal the slide with neutral gum and place in a ventilated place to dry.

[0064] The zebrafish intestinal tissue sections obtained in this example are shown in the attached Figure 1 The intestine is the main place for zebrafish to digest and absorb food. As can be seen from the picture, the intestinal wall of zebrafish is relatively thick, and the intestinal villi are clearly visible, showing staggered branches and then gradually becoming finger-shaped; goblet cells are large and round, arranged between the intestinal villi.

[0065] Example 2

[0066] In order to highlight the effect of the microwave paraffin sectioning method of the present invention, a conventional paraffin sectioning method for an animal tissue in this embodiment is used. Taking zebrafish intestinal tissue as an example, a conventional paraffin sectioning method is adopted. The experimental steps are basically the same as those in Example 1, wherein the four steps of fixation, dehydration, transparency and wax immersion do not require microwave treatment, and the time of conventional treatment is different from that of microwave treatment. For specific time, see Table 1 below.

[0067] Table 1 Comparison of experimental cycles between conventional paraffin sectioning and microwave paraffin sectioning

[0068] Production steps Conventional paraffin sectioning Microwave paraffin sectioning fixed 24h 10-20min Dehydration 12h 1h transparent 12h 30min Wax Dipping 48h 1h Embedding and sectioning 2h 2h Staining and sealing 1h 1h Total duration About 100h About 6 hours

[0069] Zebrafish intestinal tissue sections obtained by conventional paraffin sectioning method are shown in the attached Figure 2 As can be seen from the above table, it takes about 100 hours to prepare zebrafish intestinal tissue slices using the conventional paraffin sectioning method, while it takes about 100 hours to prepare zebrafish intestinal tissue slices using the microwave paraffin sectioning method of the present invention. Figure 1 and Figure 2 It can be seen that the zebrafish intestinal tissue sections obtained by the two methods are basically the same, and the tissues are not deformed or broken. However, there are slight differences in details. Figure 1 The intestinal tissue structure is intact and the intestinal tissue morphology is clearer, which shows that shortening the processing time has less impact on the tissue structure and can better display the original morphology of the tissue structure. Figure 2 The intestinal tissue morphology in the sample is basically clear, but the goblet cells on the intestinal villi are partially detached, which is due to the long processing time of the entire section. It can be seen that the sections obtained by the microwave paraffin sectioning method of Example 1 of the present invention are clearer.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing microwave efficient paraffin sections of animal tissues, characterized in that: The steps include: (1) Dissection: remove the required animal tissues and disinfect them for later use; (2) Fixation: Place the tissue sample in a glass vial and pour in neutral formaldehyde fixative until the tissue sample is covered; take a 200 mL beaker, add 50 mL of water, and then heat the water to 50 °C. Place the glass vial containing the tissue sample in the beaker, put the beaker in a microwave oven, set the microwave oven to low heat for 20 minutes, heat for 5 minutes, maintain the water temperature at 50 °C, and take out the beaker; replace the fixative and repeat the microwave heating step once; then place the glass vial containing the tissue sample at room temperature for later use; (3) Dehydration treatment: a) Aspirate the neutral formaldehyde fixative in the glass vial and add 50% v / v ethanol solution until the tissue sample is covered; place the glass vial in a beaker, put the beaker in a microwave oven, set the microwave oven to low heat 20, heat for 5 min, maintain the water temperature at 50 ℃, take out the beaker and let it stand for 1 min; b) Aspirate the 50% v / v ethanol solution in the glass vial and add 70% v / v ethanol solution until the tissue sample is covered; put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 5 min, maintain the water temperature at 50 ℃, take out the beaker and let it stand for 1 min; c) Aspirate the 70% v / v ethanol solution in the glass vial and add 85% v / v ethanol solution until the tissue sample is covered; put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 5 min, maintain the water temperature at 50 ℃, take out the beaker and let it stand for 1 min; d) Aspirate the 85% v / v ethanol solution in the glass vial and add 90% v / v ethanol solution until the tissue sample is covered; put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 5 min, maintain the water temperature at 50 °C, take out the beaker, and let it stand for 1 min; e) Aspirate the 90% v / v ethanol solution in the glass vial and add anhydrous ethanol solution until the tissue sample is covered; place the glass vial in a beaker, place the beaker in a microwave oven, set the microwave oven to low heat 20, heat for 5 min, maintain the water temperature at 50 °C, take out the beaker, let it stand for 1 min, and prepare for subsequent treatment; (4) Transparent processing: a) Aspirate the anhydrous ethanol solution in the above glass vial, add anhydrous ethanol-xylene mixture with a volume ratio of 1:1 until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 5 min, maintain the water temperature at 50 ℃, take out the beaker, and let it stand for 1 min; b) Aspirate the anhydrous ethanol-xylene mixture in the above glass vial, add xylene solution until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 5 min, maintain the water temperature at 50 ℃, take out the beaker, and let it stand for 1 min; c) Aspirate the xylene solution in the above glass vial and add new xylene solution until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 5 min, maintain the water temperature at 50 °C, take out the beaker, let it stand for 1 min, and prepare for subsequent treatment; (5) Wax treatment a) Aspirate the xylene solution in the glass vial, add a mixture of melted paraffin and xylene preheated to 55°C in a volume ratio of 1:1 until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 10 min, maintain the water temperature at 55°C, take out the beaker, and let it stand for 1 min; b) Aspirate the paraffin-xylene mixture in the glass vial, add a pre-heated melted paraffin and xylene mixture in a volume ratio of 4:1, until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 10 min, maintain the water temperature at 55 °C, take out the beaker, and let it stand for 1 min; c) Aspirate the paraffin-xylene mixture in the glass vial, add the pre-heated melted pure paraffin solution until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 10 min, maintain the water temperature at 55 °C, take out the beaker, and let it stand for 1 min; d) Pipette out the pure paraffin solution in the above glass vial, add new pure paraffin solution that has been pre-heated and melted, until the tissue sample is covered; seal the glass vial with a sealing film, put the glass vial into a beaker, put the beaker into a microwave oven, set the microwave oven to low heat 20, heat for 10 min, maintain the water temperature at 55 °C, take out the beaker, let it stand for 1 min, and prepare for subsequent treatment; (6) Embedding treatment Pour the tissue material in the glass vial treated above into a paper box of liquid paraffin to cool; (7) Sectioning and spreading The solidified paraffin and materials were sliced ​​with a microtome, with a slice thickness of 10 μm, and then the wax strips were placed at 37 °C for expansion; (8) Staining and sealing Use 1% toluidine blue dye solution for staining, specifically: place the slide in the following solutions: xylene solution for 30 min → 1:1 anhydrous ethanol-xylene mixture for 90 s → anhydrous ethanol solution for 90 s → 95% ethanol solution for 90 s → 85% ethanol solution for 90 s → 70% ethanol solution for 90 s → 50% ethanol solution for 90 s → 30% ethanol solution for 90 s → distilled water for 90 s → 1% toluidine blue dye solution for 90 s → distilled water for 90 s → 30% ethanol solution for 90 s → 50% ethanol solution for 90 s → 70% ethanol solution for 90 s → 85% ethanol solution for 90 s → 95% ethanol solution for 90 s → anhydrous ethanol solution for 90 s→mixture of anhydrous ethanol and xylene in a volume ratio of 1:1 90 s→pure xylene solution, and finally seal the slides with neutral gum and place them in a ventilated place to dry.

2. The use of the microwave efficient paraffin sectioning method for animal tissue according to claim 1, characterized in that: The animal tissue is zebrafish intestine, mouse intestine or rabbit intestine.

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