Environment-friendly biological tissue sample preparation sleeve solution and application thereof

Abstract

The invention discloses an environment-friendly biological tissue sample preparation sleeve solution and application thereof, and belongs to the technical field of application of pathological reagents in the pathology department. In order to solve the technical problems that in the prior art, in the tissue fixing and tissue wax block preparation process, the fixing effect is not ideal, fixing liquid and transparent liquid pollute the environment and harm human health, dehydration liquid is not thorough in dehydration, subsequent wax dipping liquid permeation is affected, wax blocks cannot be sliced, and the environment is polluted. The invention provides an environment-friendly biological tissue sample preparation set solution and application thereof, the preparation set solution is composed of a stationary solution, a dehydration solution and a transparent solution, and the stationary solution is composed of 40-70 parts of a protein fixing agent, 1-10 parts of a preservative, 1-5 parts of an osmotic pressure regulator, 2-5 parts of a wetting agent, 0.1-0.5 part of an antioxidant and 15-45 parts of purified water. The prepared sleeve solution can be applied to pathological histology and animal and plant specimen preparation, including conventional pathological diagnosis, immunohistochemical diagnosis technology, molecular biological diagnosis technology and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the pathological reagent application technical field in the pathological department, and particularly relates to application of an environment-friendly biological tissue sample preparation set liquid in pathological histology and animal and plant specimen making. BACKGROUND

[0002] In the field of medical pathological diagnosis, the fixation of tissue sections is usually formaldehyde fixing solution or polyhydric alcohol combined fixing solution. Formaldehyde has strong hardening effect on tissue fixation, strong penetration and good sterilization effect, but its obvious shortcomings are that it can cause great harm to the human lung and even induce lung cancer after being inhaled. The polyhydric alcohol fixing solution has poor tissue penetration and is difficult to penetrate into the deep part of the tissue, and the fixing effect is not ideal, and the tissue is prone to shrinkage, discoloration and surface hardening. The traditional gradient ethanol dehydrating solution and the commercially available dehydrating solution composed of ethanol, isopropyl alcohol, tert-butyl alcohol, acetone and methylcyclohexane can easily absorb moisture. When the laboratory is rainy or the humidity is too high, the dehydrating solution can quickly absorb the moisture in the air, resulting in an increase in water content, and it is difficult to completely remove the water from the tissue sections, causing the tissue to become brittle. Due to poor tissue dehydration, the paraffin solution cannot penetrate into the tissue, causing the wax block to become soft, or the tissue to become hard and brittle due to "dry roasting" in the high-temperature paraffin solution, and even the dehydrated tissue shrinks and forms a gap with the wax block, which ultimately cannot be sliced, resulting in the failure to complete the medical pathological diagnosis and causing a medical accident. In addition, the tert-butyl alcohol in the dehydrating solution has strong toxicity, the acetone can corrode the dehydrating machine pipeline and cause tissue shrinkage, and the methylcyclohexane in the defatting component has strong irritancy and toxicity.

[0003] In the field, efforts have been made to improve safety, improve fixation effect, improve tissue dehydration quality, improve the quality of wax blocks and subsequent slice making, and the biological tissue sample preparation set liquid composed of environmentally friendly components is harmless or low-toxic to the human body.

[0004] The patent document CN109805001A describes a "formaldehyde-free tissue fixation and preservation liquid and its preparation method", which solves the above problems, improves the fixation effect and preservation ability, and improves the safety to the human body without using formaldehyde. However, the formaldehyde-free tissue fixation and preservation liquid provided by the scheme will appear turbidity after soaking the tissue, which will cause the loss of tissue components and thus cause pollution to other tissues, resulting in false negative or false positive phenomenon in pathological diagnosis. SUMMARY

[0005] The present application provides an environment-friendly biological tissue sample preparation set liquid and its application to solve the technical problems of unsatisfactory fixation effect in the process of tissue fixation and tissue wax block preparation, environmental pollution and harm to human health caused by fixation liquid and transparent liquid, incomplete dehydration of dehydrating solution, influence on subsequent paraffin infiltration, and inability to slice the wax block.

[0006] One of the purposes of the present application is to provide an environmentally friendly biological tissue sample preparation kit, which comprises a fixing solution, a dehydrating solution and a transparent solution.

[0007] In a preferred embodiment of the present application, the fixing solution comprises 40-70 parts of a protein fixative, 1-10 parts of a preservative, 1-5 parts of an osmotic pressure regulator, 2-5 parts of a wetting agent, 0.1-0.5 parts of an antioxidant and 15-45 parts of purified water.

[0008] In a preferred embodiment of the present application, the protein fixative comprises 40-70 parts of ethanol, 0.1-0.3 parts of dimethyl sulfoxide, 0.1-0.3 parts of carbonized diimide and 2-10 parts of propylene glycol.

[0009] In a preferred embodiment of the present application, the preservative comprises 0.1-0.3 parts of dodecyl dimethyl benzyl ammonium chloride, 0.1-0.3 parts of bisquill dimethyl ammonium chloride and 40-70 parts of ethanol.

[0010] In a preferred embodiment of the present application, the osmotic pressure regulator comprises 0.1-0.3 parts of thione and 0.05-0.2 parts of sucrose; the wetting agent is 1-8 parts of polyethylene glycol; and the antioxidant is 0.01-0.05 parts of vitamin E.

[0011] In a preferred embodiment of the present application, the dehydrating solution comprises 90-98 parts of ethanol, 0.1-0.3 parts of thione and 1.7-10 parts of purified water for dehydrating I and II; 10-30 parts of ethanol, 50-90 parts of n-propanol and 0.05-0.3 parts of thione for dehydrating III and IV; 30-70 parts of n-propanol, 20-40 parts of isododecane, 4-10 parts of isopentadecane, 0.5-4 parts of isomyristic acid and 0.5-3 parts of thione for dehydrating V and VI.

[0012] In a preferred embodiment of the present application, the transparent solution comprises 50-90 parts of isododecane, 10-40 parts of isopentadecane, 5-10 parts of isomyristic acid and 5-10 parts of isohexadecane.

[0013] Another purpose of the present application is to provide a use method of the above-mentioned environmentally friendly biological tissue preparation kit, which comprises the following steps:

[0014] S1: sampling a biological tissue sample with a size of 1.5 cm x 1.5 cm x 0.2-0.4 cm to obtain a treatment sample;

[0015] S2: the processing sample obtained in S1 can be subjected to tissue processing by manual or automatic tissue dehydrator, and the procedure is as follows: the processing time of the fixing solution is 2 h for the first time and 2-2.5 h for the second time; the processing time of the dehydrating solution I-VI is 1 h; the processing time of the transparent solution is 1 h for the first time and 1 h for the second time.

[0016] In a preferred embodiment of the present application, when the processing sample in S1 is a gastrointestinal biopsy sample, the processing time of each reagent in the tissue processing procedure is reduced by 1 / 2; when the processing sample is a liver or kidney or puncture biopsy tissue sample, the processing time of each reagent in the tissue processing procedure is reduced by 2 / 3.

[0017] The third object of the present application is to provide the application of the above-mentioned environmentally friendly biological tissue preparation set of solutions in pathological histology and animal and plant specimen preparation.

[0018] The present application has the following advantages:

[0019] The preparation set of solutions provided by the present application has the following advantages compared with the scheme described in patent document CN109805001A and the traditional set of solutions:

[0020] (1) The present application activates the carboxyl group in the protein or polypeptide in the tissue by adding carbonized diimine, so that it forms an amide bond with the amino group or an ester with ethanol / propylene glycol, thereby making the protein lose activity and achieving the effect of tissue fixation; compared with 2-methyl-1,2-benzisothiazol-3-ketone, carbonized diimine has a more moderate fixation effect and will not cause damage to the pretreated tissue or destroy its structure, and from the appearance, the tissue fixed with carbonized diimine as the effective fixing component is complete without loss, and the fixing solution after soaking the tissue is clear and not turbid; while the fixing solution with 2-methyl-1,2-benzisothiazol-3-ketone as the effective component will appear turbid after soaking the tissue.

[0021] (2) The present application uses thione as an osmotic pressure regulator, which has stronger penetration ability, and by acting on the stratum corneum of human and animal tissues, the intercellular space of the surface cells is increased, and the defense ability is decreased, so that the tissue fixation component can quickly enter the tissue cells and reach the target, and at the same time, it cooperates with dodecyl dimethyl benzyl ammonium chloride, dipentaerythritol dimethyl ammonium chloride and sucrose to achieve the purpose of rapid fixation of the tissue.

[0022] (3) The fixed liquid effective component provided by the application not only has the sterilization and preservation function, but also has the function of stripping the mucus on the surface of the tissue to affect the tissue fixation and dyeing, so as to increase the penetration ability of the fixed liquid to the tissue, and also has a certain degreasing function, thereby providing sufficient conditions for preparing high-quality pathological wax blocks / slices; compared with the conventional formaldehyde fixed liquid, the fixed liquid provided by the application has higher safety and smaller harm to the human body, does not pollute the environment, and is more friendly to the environment; compared with the polyhydric alcohol fixed liquid, the fixed liquid has stronger permeability, is easier to penetrate into the inside of the tissue to realize rapid fixation, and the tissue is not shrunk, discolored or hardened on the surface.

[0023] (4) Compared with the traditional gradient ethanol dehydration liquid or the dehydration liquid composed of components such as commercially available ethanol, isopropanol, tert-butanol, acetone or methylcyclohexane, the dehydration liquid provided by the application is not easy to absorb moisture, has a longer service life, smaller irritation, and less obvious tissue shrinkage, the wax block tissue treated by the dehydration liquid is moderate in softness and toughness, has full tenacity, has no corrosion effect on the pipeline system of the dehydration machine, and has less influence on the subsequent processing of other specimens; the dehydration liquid provided by the application has stronger component fusion ability, the transparent liquid has good compatibility with mineral oil or xylene as a solvent, has good optical properties, and the prepared slice specimen is clear and transparent under a microscope. DETAILED DESCRIPTION

[0024] Those skilled in the art can improve the process parameters according to the content herein. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the application. The method and application of the application have been described by the preferred embodiments, and the related personnel can obviously modify or appropriately change and combine the method and application described herein to realize and apply the technology of the application without departing from the content and scope of the application.

[0025] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be further described in detail below in combination with specific embodiments. The experimental methods used in the following examples are conventional methods, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art, which can be obtained by commercial channels.

[0026] Example 1

[0027] (1) 5 parts by volume of propylene glycol, 0.2 parts by volume of carbon diimide, 0.2 parts by volume of dimethyl sulfoxide, 8 parts by volume of polyethylene glycol, 0.2 parts by volume of thione and 0.03 parts by volume of vitamin E are sequentially dissolved in 55 parts by volume of ethanol, mixed uniformly as A liquid for standby; 0.1 parts by volume of sucrose, 0.2 parts by volume of dodecyl dimethyl benzyl ammonium chloride and 0.2 parts by volume of dipentaerythritol dimethyl ammonium chloride are sequentially dissolved in 33.87 parts by volume of pure water, stirred uniformly as B liquid for standby; finally, the B liquid is uniformly mixed in the A liquid to obtain a fixing liquid;

[0028] (2) 95 parts by volume of ethanol, 0.2 parts by volume of thione and 4.8 parts by volume of pure water are mixed uniformly to obtain dehydration liquids I and II, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0029] 20 parts by volume of ethanol, 80 parts by volume of n-propanol, 0.2 parts by volume of thione are mixed uniformly to obtain dehydration liquids III and IV, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0030] 60 parts by volume of n-propanol, 30 parts by volume of isododecane, 6 parts by volume of isopentadecane, 3 parts by volume of isomyristic acid and 1 part by volume of thione are mixed uniformly to obtain dehydration liquids V and VI, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0031] (3) 75 parts by volume of isododecane, 25 parts by volume of isopentadecane, 8 parts by volume of isomyristic acid and 8 parts by volume of isohexadecane are mixed uniformly to obtain a transparent liquid.

[0032] Example 2

[0033] (1) 10 parts by volume of propylene glycol, 0.1 parts by volume of carbon diimide, 0.1 parts by volume of dimethyl sulfoxide, 8 parts by volume of polyethylene glycol, 0.1 parts by volume of thione and 0.01 parts by volume of vitamin E are sequentially dissolved in 40 parts by volume of ethanol, mixed uniformly as A liquid for standby; 0.05 parts by volume of sucrose, 0.1 parts by volume of dodecyl dimethyl benzyl ammonium chloride and 0.1 parts by volume of dipentaerythritol dimethyl ammonium chloride are sequentially dissolved in 41.45 parts by volume of pure water, stirred uniformly as B liquid for standby; finally, the B liquid is uniformly mixed in the A liquid to obtain a fixing liquid;

[0034] (2) 90 parts by volume of ethanol, 0.3 parts by volume of thione and 9.7 parts by volume of pure water are mixed uniformly to obtain dehydration liquids I and II, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0035] Mixing 10 parts by volume of ethanol, 89.8 parts by volume of n-propanol, and 0.3 part by volume of thixotropic agent uniformly, dehydrating liquids III and IV are obtained, which are immediately filled to avoid absorbing moisture in the air for a long time to affect the dehydration effect;

[0036] Mixing 30 parts by volume of n-propanol, 40 parts by volume of isododecane, 10 parts by volume of isopentadecane, 4 parts by volume of isohexadecane, and 3 parts by volume of thixotropic agent uniformly, dehydrating liquids V and VI are obtained, which are immediately filled to avoid absorbing moisture in the air for a long time to affect the dehydration effect;

[0037] (3) Mixing 50 parts by volume of isododecane, 40 parts by volume of isopentadecane, 10 parts by volume of isohexadecane, and 10 parts by volume of isohexadecane uniformly, a transparent liquid is obtained.

[0038] Example 3

[0039] (1) Dissolving 7 parts by volume of propylene glycol, 0.25 part by volume of carbon diimide, 0.25 part by volume of dimethyl sulfoxide, 7 parts by volume of polyethylene glycol, 0.25 part by volume of thixotropic agent, and 0.02 part by volume of vitamin E in 50 parts by volume of ethanol in sequence, and mixing uniformly to obtain A liquid for standby; dissolving 0.1 part by volume of sucrose, 0.2 part by volume of dodecyl dimethyl benzyl ammonium chloride, and 0.2 part by volume of dipentaerythritol dimethyl ammonium chloride in 34.73 parts by volume of pure water in sequence, and stirring uniformly to obtain B liquid for standby; finally, mixing B liquid uniformly in A liquid to obtain a fixing liquid;

[0040] (2) Mixing 93 parts by volume of ethanol, 0.2 part by volume of thixotropic agent, and 6.8 parts by volume of pure water uniformly, dehydrating liquids I and II are obtained, which are immediately filled to avoid absorbing moisture in the air for a long time to affect the dehydration effect;

[0041] Mixing 15 parts by volume of ethanol, 84.8 parts by volume of n-propanol, and 0.2 part by volume of thixotropic agent uniformly, dehydrating liquids III and IV are obtained, which are immediately filled to avoid absorbing moisture in the air for a long time to affect the dehydration effect;

[0042] Mixing 50 parts by volume of n-propanol, 25 parts by volume of isododecane, 8 parts by volume of isopentadecane, 4 parts by volume of isohexadecane, and 3 parts by volume of thixotropic agent uniformly, dehydrating liquids V and VI are obtained, which are immediately filled to avoid absorbing moisture in the air for a long time to affect the dehydration effect;

[0043] (3) Mixing 70 parts by volume of isododecane, 20 parts by volume of isopentadecane, 10 parts by volume of isohexadecane, and 10 parts by volume of isohexadecane uniformly, a transparent liquid is obtained.

[0044] Example 4

[0045] (1) 3 parts by volume of propylene glycol, 0.15 parts by volume of carbon diimide, 0.15 parts by volume of dimethyl sulfoxide, 3 parts by volume of polyethylene glycol, 0.15 parts by volume of thione and 0.02 parts by volume of vitamin E are sequentially dissolved in 60 parts by volume of ethanol, mixed uniformly as A liquid for standby; 0.08 parts by volume of sucrose, 0.15 parts by volume of dodecyl dimethyl benzyl ammonium chloride and 0.15 parts by volume of dipentadecyl dimethyl ammonium chloride are sequentially dissolved in 33.15 parts by volume of pure water, stirred uniformly as B liquid for standby; finally, the B liquid is uniformly mixed in the A liquid to obtain a fixing liquid;

[0046] (2) 97 parts by volume of ethanol, 0.25 parts by volume of thione and 2.75 parts by volume of pure water are mixed uniformly to obtain dehydration liquids I and II, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0047] 25 parts by volume of ethanol, 74.8 parts by volume of n-propanol, 0.2 parts by volume of thione are mixed uniformly to obtain dehydration liquids III and IV, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0048] 65 parts by volume of n-propanol, 25 parts by volume of isododecane, 4 parts by volume of isopentadecane, 4 parts by volume of isomyristic acid and 2 parts by volume of thione are mixed uniformly to obtain dehydration liquids V and VI, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0049] (3) 85 parts by volume of isododecane, 20 parts by volume of isopentadecane, 7 parts by volume of isomyristic acid and 7 parts by volume of isohexadecane are mixed uniformly to obtain a transparent liquid.

[0050] Example 5

[0051] (1) 2 parts by volume of propylene glycol, 0.1 parts by volume of carbon diimide, 0.1 parts by volume of dimethyl sulfoxide, 1 part by volume of polyethylene glycol, 0.1 parts by volume of thione and 0.01 parts by volume of vitamin E are sequentially dissolved in 70 parts by volume of ethanol, mixed uniformly as A liquid for standby; 0.05 parts by volume of sucrose, 0.1 parts by volume of dodecyl dimethyl benzyl ammonium chloride and 0.1 parts by volume of dipentadecyl dimethyl ammonium chloride are sequentially dissolved in 26.44 parts by volume of pure water, stirred uniformly as B liquid for standby; finally, the B liquid is uniformly mixed in the A liquid to obtain a fixing liquid;

[0052] (2) 97 parts by volume of ethanol, 0.2 parts by volume of thione and 1.8 parts by volume of pure water are mixed uniformly to obtain dehydration liquids I and II, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0053] Mixing 30 parts by volume of ethanol, 69.8 parts by volume of n-propanol, and 0.2 parts by volume of thione uniformly, dehydrating liquids III and IV are obtained, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0054] Mixing 70 parts by volume of n-propanol, 25 parts by volume of isododecane, 4 parts by volume of isopentadecane, 0.5 parts by volume of isomyristic acid, and 1.5 parts by volume of thione uniformly, dehydrating liquids V and VI are obtained, which are immediately filled to avoid long-time contact with air to absorb moisture in the air and affect the dehydration effect;

[0055] (3) Mixing 90 parts by volume of isododecane, 10 parts by volume of isopentadecane, 5 parts by volume of isomyristic acid, and 5 parts by volume of isohexadecane uniformly, a transparent liquid is obtained.

[0056] Comparative Example 1

[0057] The conventional commercial neutral formaldehyde fixing solution, ethanol gradient dehydrating liquid, and xylene transparent liquid are used in this comparative example.

[0058] Comparative Example 2

[0059] According to the tissue fixing solution preparation method in the patent “A formaldehyde-free tissue fixing and preserving solution and a preparation method thereof”, 2-methyl-1,2-benzisothiazolin-3-one (MBIT) 2 kg, dimethyl sulfoxide 7 kg, and azone 3 kg are melted in 55 kg of ethanol to obtain a protein fixing agent; then 5-chloro-2-methyl-4-isothiazolin-3-one 0.06 kg and 2-methyl-4-isothiazolin-3-one 0.02 kg, sodium nitrate 0.5 kg, sodium chloride 1 kg, sucrose 1 kg, and 0.2 kg of vitamin C are melted in 10.22 kg of water, and then the two mixed liquids obtained above are mixed and then a humectant (isopropyl alcohol 5 kg, propylene glycol 5 kg, and polyethylene glycol 10 g) is added, and stirred uniformly to obtain a fixing and preserving solution; wherein the ethanol gradient dehydrating liquid and the xylene transparent liquid are conventional commercial reagents.

[0060] I. Comparison of the effects of fixing solutions

[0061] The present application uses the fixing solution prepared in Examples 1-5, the commercially available conventional neutral formaldehyde fixing solution in Comparative Example 1 and the fixing solution prepared in Comparative Example 2 to respectively fix rat liver, rectum and kidney, and the specific steps are as follows: firstly, sample rat liver, rectum and kidney, and the sample size is 1.5 cm x 1.5 cm x 0.2-0.4 cm, and the limit value should be less than the volume that can be accommodated by the tissue embedding box. Excessive extrusion and filling of the tissue embedding box can affect the effective contact of the reagent and the tissue specimen, and reduce the specimen processing quality. Subsequently, the above-mentioned tissues or tissue sections are respectively immersed in 5 times the volume of the fixing solution, the container is closed, the indoor temperature is kept at 25 DEG C, the time required for each group of specimens to complete the fixation is observed and recorded, the sample color and hardness, deformation condition are observed and recorded, and the clarity of the fixing solution is detected and recorded, and the results are shown in Table 1.

[0062] Table 1 Comparison of fixing solution on small tissue specimen fixation

[0063]

[0064] The fixing solution effect comparison results are shown in Table 1, the tissue sample fixation time of the best ratio of the solution obtained in Example 1 is less than that of the comparative examples, and the tissue sample fixation time of the solutions prepared in Examples 2-5 is also shorter than that of Comparative Examples 1-2. Through comparison of the specimen hardness and deformation, it is found that the kidney sample after fixation of Comparative Example 1 presents the characteristics of hard and brittle, and the tissue samples after fixation of the fixing solutions prepared in the present application have good specimen hardness and deformation. Through comparison of the clarity of the fixing solution after fixation, it is found that the fixing solutions of Examples 1-2 are clear, and the fixing solution of Example 3 appears slightly turbid, which is speculated to be caused by the high content and proportion of the penetration enhancer such as thione, sucrose, dodecyl dimethyl benzyl ammonium chloride and dipentaerythritol dimethyl ammonium chloride, thereby causing the dissolution of intracellular small molecular components of the tissue, resulting in the appearance of the fixing solution slightly turbid. Comparative Examples 1-2 all appear slightly turbid and turbid. In summary, the biological tissue fixing solution provided by the present application has good fixing effect in a short time, and the ratio of the components of the fixing solution in Example 1 is the best ratio, and the fixing solution prepared thereby has the best comprehensive fixing effect.

[0065] II. Effect of the solution and preparation of the cutting block

[0066] The present application uses the fixing solution prepared in Examples 1-5, the commercially available conventional neutral formaldehyde fixing solution in Comparative Example 1 and the fixing solution prepared in Comparative Example 2 to fix, dehydrate, transparentize and wax-impregnate rat liver respectively, and the fixing solution is comprehensively judged according to the changes of reagents during the treatment process and the quality of the wax block. The dehydrator is a full-automatic dehydrator of the model of Leica ASP300S, the treatment time and conditions of reagents other than those of the present application are used according to the requirements of the seller's manual, the size of the sample is 1.5 cm x 1.5 cm x 0.2-0.4 cm, the limit value should be less than the volume that can be accommodated by the tissue embedding box, excessive extrusion and filling of the tissue embedding box will affect the effective contact of the reagent with the tissue sample and reduce the treatment quality of the sample. The comparison results are shown in Table 2.

[0067] Table 2 Comparison of comprehensive treatment of rat liver

[0068]

[0069] In the process of preparing tissue wax blocks, the dehydration step consists of 6 parts. In the treatment of dehydration solution I and II, most of the water in the tissue sample has been replaced. In the treatment of dehydration solution III, IV, V and VI, most of the water is absorbed from the air during the contact with the air. Methanol and ethanol are easily hygroscopic, while n-propanol is not. Therefore, n-propanol is added to the dehydration solution to avoid the absorption of water from the air and affect the effect of the fixing solution. However, n-propanol has a larger molecule than methanol and ethanol, and is relatively difficult to penetrate the cell cuticle and cell membrane to replace the internal water. However, the addition of thione osmotic factor and a small amount of other reagents promotes the effect of n-propanol in replacing the internal water, so that the dehydration solution of the present application has a good dehydration effect. As can be seen from Table 2, although the service life of the dehydration solution in Examples 2-3 is longer than that in Example 1, the dehydration efficiency is lower than that in Example 1. Although the dehydration efficiency of the dehydration solution in Examples 4-5 is higher than that in Example 1, the service life is significantly shorter than that of the dehydration solution in Example 1 due to the increase of the easily hygroscopic component. The dehydration effect and efficiency of Comparative Examples 1-2 are not as good as those of Examples 1-5, which proves that the ratio of the components in Example 1 is the best and has the best effect. It is further proved that the amount of n-propanol added has a certain limit and needs to be added within the best ratio range to achieve the best dehydration effect in the shortest time.

[0070] When the water content of the dehydrating solution exceeds 5%, the clarity of the subsequent transparent solution and the hardness of the wax block / sample will be affected, because the hardness of the wax block / sample is low, which brings great difficulty to the preparation of the wax block section, and even because of the excessive water content of the tissue, the volatilization of water at high temperature causes the tissue to shrink, and the air gap between the wax block and the tissue is easy to produce, so that the wax block section is discontinuous or incomplete, and if the dehydration is not clean, even if the wax-sealed tissue does not shrink, the toughness of the tissue is not enough, which causes the wax block section to break or be continuous. Therefore, from Tables 1-2, it can be seen that the preparation of the solution obtained in Examples 1-5 is better than that of the preparation solution obtained in Comparative Examples 1-2 in terms of fixation, dehydration effect, preparation of tissue section quantity and preparation of wax block quality, and through fine comparison and analysis of Examples 1-5, it is found that the preparation solution obtained in Example 1 has the best effect on the preparation of biological tissue section.

[0071] The content not described in detail in the specification of the present application is the technology known to those skilled in the art. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. An environmentally friendly biological tissue preparation solution, characterized in that, The preparation solution consists of the following components: fixative, dehydrating solution, and transparent solution.

2. The preparation solution according to claim 1, characterized in that, The fixative consists of the following components: 40-70 parts protein fixative, 1-10 parts preservative, 1-5 parts osmotic pressure regulator, 2-5 parts wetting agent, 0.1-0.5 parts antioxidant, and 15-45 parts purified water.

3. The preparation solution according to claim 2, characterized in that, The protein fixative is composed of the following components: 40-70 parts ethanol, 0.1-0.3 parts dimethyl sulfoxide, 0.1-0.3 parts carbodiimide, and 2-10 parts propylene glycol.

4. The preparation solution according to claim 2, characterized in that, The preservative is composed of the following components: 0.1-0.3 parts of dodecyl dimethyl benzyl ammonium chloride, 0.1-0.3 parts of didecyl dimethyl ammonium chloride, and 40-70 parts of ethanol.

5. The preparation solution according to claim 2, characterized in that, The osmotic pressure regulator is composed of the following components: 0.1-0.3 parts of thiazoline and 0.05-0.2 parts of sucrose; the wetting agent is 1-8 parts of polyethylene glycol; and the antioxidant is 0.01-0.05 parts of vitamin E.

6. The preparation solution according to claim 1, characterized in that, The dehydration solution is composed of the following components: Dehydration I and II consist of 90-98 parts ethanol, 0.1-0.3 parts thiatone, and 1.7-10 parts purified water; Dehydration III and IV consist of 10-30 parts ethanol, 50-90 parts n-propanol, and 0.05-0.3 parts thiatone; Dehydration V and VI consist of 30-70 parts n-propanol, 20-40 parts isododecane, 4-10 parts isopentadecanane, 0.5-4 parts isoentanecanane, and 0.5-3 parts thiatone.

7. The preparation solution according to claim 1, characterized in that, The transparent liquid is composed of the following components: 50-90 parts of isododecane, 10-40 parts of isopentadecanane, 5-10 parts of isoentanecanane, and 5-10 parts of isohexadecane.

8. The method of using the environmentally friendly biological tissue preparation solution according to any one of claims 1 to 7, characterized in that, The method of use includes the following steps: S1: Take samples of biological tissue, with a sample size of 1.5cm × 1.5cm × 0.2-0.4cm, to obtain processed samples; S2: The processed samples obtained in S1 can be processed manually or automatically using a tissue dehydrator. The procedure is as follows: the treatment time for fixative 1 is 2 hours, and the treatment time for 2-2.5 hours; the treatment time for dehydration solutions I-VI is 1 hour each; the treatment time for clearing solution 1 is 1 hour, and the treatment time for 2 hours is 1 hour.

9. The method of use according to claim 8, characterized in that, When the sample to be processed in S1 is a gastrointestinal biopsy sample, the processing time of each reagent in the tissue processing procedure is reduced by 1 / 2; when the sample to be processed is a liver, kidney, or puncture biopsy tissue sample, the processing time of each reagent in the tissue processing procedure is reduced by 2 / 3.

10. The application of the environmentally friendly biological tissue preparation solution according to any one of claims 1-7 in pathological histology and animal and plant specimen preparation.

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