An environmentally friendly dewaxing transparent reagent and its preparation method and application

Abstract

The present invention provides an environmentally friendly dewaxing transparent reagent and its preparation method and application, the environmentally friendly dewaxing transparent reagent includes an initiator, a wax dissolving agent, a transparent agent and a solution modifier; the initiator includes normal paraffin; the wax dissolving agent includes isoparaffin; the transparent agent includes any one or a combination of at least two of tert-butyl alcohol, PEG methacrylate, N,N,N,N-tetrakis (2-hydroxypropyl) ethylenediamine, benzyl benzoate, polyethylene glycol, benzyl alcohol or diphenyl ether. The environmentally friendly dewaxing transparent reagent is used for film-making process, the environmentally friendly dewaxing transparent reagent has stable wax dissolving, fast wax dissolving speed, low volatility, low odor, high ignition point, and the dewaxing transparent reagent can reduce the tissue refractive index, so that the slice is more uniform and transparent, and the slice structure is clear. The raw material of the dewaxing transparent reagent is a non-toxic and safe product, which is safe and convenient to use.

Description

Technical Field

[0001] The invention belongs to the technical field of pathological tissue sections, and in particular relates to an environmentally friendly dewaxing transparent reagent and a preparation method and application thereof. Background Art

[0002] Conventional paraffin-embedded tissue sections (conventional sections) are the most widely used technique in conventional histological section preparation. They are often used in pathology to study, observe and judge the morphological changes of cell tissues. Conventional sections combined with immunological techniques can be used to qualitatively and positionally observe and study macromolecules such as polypeptides and proteins in cell tissues in tissue sections. Conventional sections combined with fluorescence in situ hybridization (FISH) can be used for gene localization and status analysis in cells. They have been widely used in research in various disciplines.

[0003] Formalin-fixed paraffin-embedded tissue is the most commonly used sample preservation method in the pathology department. The preserved samples are stable and the storage conditions are simple. They can be kept in hospital archives for several years to decades.

[0004] The primary task of pathological technology is to produce high-quality sections. In the HE (Hematoxylin-Eosin) conventional paraffin sectioning technology, tissue transparency, section dewaxing, transparency and sealing agent preparation are inseparable from the use of transparent dewaxing agents. The transparency and dewaxing effects directly affect the quality of paraffin sectioning.

[0005] Transparent dewaxing reagent is an organic solvent that must be used in pathological slide preparation. In the process of conventional tissue paraffin slide preparation, xylene is often used as a transparent dewaxing agent. Xylene as a transparent dewaxing agent has the advantages of thorough dewaxing, strong transparency ability and short time, but it is also easy to shrink and brittle pathological tissues. The time for transparent treatment is different for different tissues, and the operating conditions are not easy to master. In addition, xylene is a moderately toxic chemical reagent, which will cause damage to the health of technicians who work in a xylene environment for a long time. Xylene is a Class 3 carcinogen recognized by the WHO and will pollute the environment. In addition, xylene is highly flammable and volatile. Xylene has a low boiling point (143℃~145℃) and a flash point of 31℃. Therefore, the use of substitutes for xylene is an inevitable trend.

[0006] Currently, the dewaxing agents reported in the literature that can replace xylene include vegetable oils, terpenes, and alkanes, etc. Each substitute can meet different application requirements.

[0007] Vegetable oils mainly include olive oil, coconut oil and peanut oil. The transparency effect of vegetable oils is comparable to that of xylene, and has less effect on tissue shrinkage. However, vegetable oils have a higher viscosity and contain saturated fatty acids. When the temperature drops below a certain critical point, turbidity or even precipitation will occur.

[0008] Terpene is the general name for a series of terpene compounds, which are olefin compounds with a molecular formula of integer multiples of isoprene. Terpene is a class of natural hydrocarbons widely found in plants, and can be obtained from many plants, especially conifers, including turpentine, d-limonene, bergamot oil, cedar oil, clove oil or oregano oil, etc. Among them, turpentine and d-limonene have been used in dewaxing agents, but such reagents have a strong odor and are not convenient to use.

[0009] Alkanes include petroleum derivatives such as gasoline, naphtha or dry cleaning solvents, which are a relatively good substitute for xylene. Some alkanes have a lighter odor, are not very oily, and have a low level of hazard. Most dewaxing and transparent products on the market are based on alkanes or are a combination of formulas.

[0010] In addition, paraffin itself can also be used as a dewaxing agent. A more effective solution is to use a mixed mixture of mineral oil and isopropanol for dewaxing. However, this method requires a higher temperature to ensure thorough dewaxing.

[0011] At present, clinical hospitals mostly use tissue dehydration and embedding equipment for pathological tissue embedding, which reduces the direct contact between medical staff and xylene. However, immunohistochemistry and FISH preparation are still mainly manual operations. Therefore, it is of great application value to develop a wax-dissolving, low-volatility, low-odor, high-ignition-point dewaxing and transparent reagent for the preparation process. Summary of the invention

[0012] The purpose of the present invention is to provide an environmentally friendly dewaxing transparent reagent and a preparation method and application thereof. The environmentally friendly dewaxing transparent reagent is used in the film making process. The environmentally friendly dewaxing transparent reagent has stable wax dissolution, fast wax dissolution speed, low volatility, low odor, and high ignition point. The dewaxing transparent reagent can reduce the refractive index of the tissue, making the slices more uniform and transparent, and the slice structure clear. The raw materials of the dewaxing transparent reagent are non-toxic and safe products, and the use is safe and convenient.

[0013] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0014] In a first aspect, the present invention provides an environmentally friendly dewaxing and transparent reagent, wherein the environmentally friendly dewaxing and transparent reagent comprises an initiator, a wax dissolving agent, a transparent agent and a solution improver;

[0015] The initiator includes normal paraffin;

[0016] The wax dissolving agent includes isoparaffin;

[0017] The transparent agent includes any one of tert-butyl alcohol, PEG methacrylate, N,N,N,N-tetrakis(2-hydroxypropyl)ethylenediamine, benzyl benzoate, polyethylene glycol, benzyl alcohol or diphenyl ether, or a combination of at least two thereof.

[0018] In the present invention, the dewaxing and transparent reagent can be miscible with paraffin and ethanol, and can make the slices more uniform and transparent by reducing the tissue refractive index, and the slice structure is clear. The raw materials of the dewaxing and transparent reagent are non-toxic and safe products, which are easy to use and can be used in general laboratories without the need to be used in a fume hood.

[0019] Preferably, the weight ratio of the initiator to the wax dissolving agent in the environmentally friendly dewaxing transparent reagent is (1-4):(2-15), for example, it can be 1:2, 1:5, 1:10, 2:5, 3:10, 1:12, 1:15 or 4:15, etc.

[0020] In the present invention, the isoparaffin reacts slowly with paraffin and is almost ineffective in dissolving wax at room temperature; the normal paraffin cannot dissolve wax at room temperature, but can dissolve wax by heating, and can dissolve wax by heating at 56°C for 2 minutes, but the dissolving wax is unstable, and as the temperature decreases, the paraffin reprecipitates, becomes a turbid solution, and even solidifies. Adding isoparaffin to normal paraffin can dewax at room temperature, and the solution is clear and not turbid.

[0021] Preferably, the environmentally friendly dewaxing and transparent reagent comprises, by weight: 1 to 4 parts of an initiator, 2 to 15 parts of a wax dissolving agent, 1 to 3 parts of a transparent agent, and 0.5 to 4 parts of a solution modifier.

[0022] In the present invention, the weight proportion of the initiator is 1 to 4 parts, for example, 1 part, 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts or 4 parts.

[0023] In the present invention, the weight proportion of the wax solvent is 2 to 15 parts, for example, it can be 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts or 15 parts.

[0024] In the present invention, the weight proportion of the transparent agent is 1 to 3 parts, for example, 1 part, 1.5 parts, 2 parts, 2.5 parts or 3 parts.

[0025] In the present invention, the weight proportion of the solution modifier is 0.5 to 4 parts, for example, 0.5 parts, 1 parts, 1.2 parts, 1.5 parts, 2 parts, 2.5 parts, 3 parts or 4 parts.

[0026] Preferably, the environmentally friendly dewaxing and transparent reagent comprises, by weight: 1 to 2 parts of an initiator, 5 to 12 parts of a wax dissolving agent, 1 to 2 parts of a transparent agent, and 0.5 to 2 parts of a solution modifier.

[0027] Preferably, the initiator includes any one of n-heptane, n-octane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane or n-hexadecane, or a combination of at least two thereof.

[0028] Preferably, the wax dissolving agent includes any one of isodecane, isoundecane, isododecane, isotridecane, isotetradecane, isopentadecane, isohexadecane, isoheptadecane, isooctadecane, isononadecane and isoicosane, or a combination of at least two of them.

[0029] In the present invention, the initiator is a C7-C16 normal chain alkane, and the wax dissolving agent is a C10-C20 isoalkane. The combined use of normal chain alkane and isoalkane can increase the effect of wax dissolving and dewaxing. At the same time, the volatility is low, the use is convenient, and the wax dissolving and dewaxing effect is good.

[0030] Preferably, the solution modifier comprises 7 # ~100 # White oil, for example, can be 7 # White oil, 10 # White oil, 15 # White oil, 20 # White oil, 32 # White oil, 40 # White oil, 50 # White oil, 60 # White oil, 70 # White oil, 80 # White oil or 100 # White oil, etc.

[0031] In the present invention, the surface tension of the isoparaffin is low, and it is easy to flow down along the edge of the container when pouring, which is inconvenient to use. By adding white oil to adjust the viscosity of the solution, the surface tension of the liquid is adjusted, making it convenient to pour. When the grade of the white oil added is larger (the viscosity of the white oil is larger), the addition amount is reduced, and when the grade of the white oil added is smaller (the viscosity of the white oil is smaller), the addition amount is increased. The surface tension of the liquid is adjusted by changing the addition amount.

[0032] Preferably, the wax dissolving agent includes any one of isodecane, isoundecane, isododecane, isotridecane, isotetradecane, isopentadecane or isohexadecane, or a combination of at least two thereof.

[0033] In the present invention, the greater the carbon content of the isoparaffin in the wax solvent, the lower its volatility, which is more conducive to use; the greater the carbon content, the worse its miscibility with ethanol, and the more initiators are needed to be used in conjunction with it.

[0034] Preferably, the clarifying agent comprises tert-butyl alcohol and / or benzyl benzoate.

[0035] In the present invention, by adding a transparent agent, the tissue section can be made more transparent, the solution after the wax is dissolved is stable, and the dissolved paraffin wax does not precipitate.

[0036] In a second aspect, the present invention provides a method for preparing the environmentally friendly dewaxing transparent reagent according to the first aspect, the preparation method comprising the following steps: mixing an initiator, a wax dissolving agent, a transparent agent and a solution modifier to obtain the environmentally friendly dewaxing transparent reagent.

[0037] Preferably, the mixing temperature is 20-25°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C.

[0038] Preferably, the mixing method includes electric stirring and / or magnetic stirring.

[0039] Preferably, the rotation speed of the electric stirring is 200-1000 rpm, for example, it can be 200 rpm, 300 rpm, 500 rpm, 800 rpm or 1000 rpm, etc., and the time of the electric stirring is 1-30 min, for example, it can be 1 min, 2 min, 5 min, 10 min, 15 min, 20 min, 25 min or 30 min, etc.

[0040] Preferably, the rotation speed of the magnetic stirring is 100-500 rpm, for example, it can be 100 rpm, 200 rpm, 300 rpm, 400 rpm or 500 rpm, etc., and the time of the magnetic stirring is 3-100 min, for example, it can be 3 min, 5 min, 15 min, 20 min, 40 min, 60 min, 80 min or 100 min, etc.

[0041] As a preferred technical solution of the present invention, the preparation method of the environmentally friendly dewaxing transparent reagent comprises the following steps: stirring the initiator, the wax dissolving agent, the transparent agent and the solution modifier by weight at 20 to 25° C. for 1 to 30 minutes at a speed of 200 to 1000 rpm to obtain the environmentally friendly dewaxing transparent reagent;

[0042] Alternatively, the initiator, wax dissolving agent, transparent agent and solution modifier are magnetically stirred at 20-25° C. for 3-100 minutes at a rotation speed of 100-500 rpm according to weight proportions to obtain the environmentally friendly dewaxing transparent agent.

[0043] In a third aspect, the present invention provides a method for using the environmentally friendly dewaxing transparent reagent according to the first aspect, the method comprising the following steps:

[0044] (1) taking a paraffin-embedded section sample, baking the section and then immersing the section in the environmentally friendly dewaxing and transparent reagent;

[0045] (2) soaking the slice sample soaked in step (1) in anhydrous ethanol;

[0046] (3) The sliced ​​samples soaked in step (2) are sequentially soaked in anhydrous ethanol, 85-90 vol% ethanol, 65-70 vol% ethanol and purified water.

[0047] In the present invention, 85-90 vol% ethanol is used for soaking the slice sample, for example, it can be 85 vol% ethanol, 86 vol% ethanol, 87 vol% ethanol, 88 vol% ethanol, 89 vol% ethanol or 90 vol% ethanol, etc.; the 65-70 vol% ethanol can be, for example, 65 vol% ethanol, 66 vol% ethanol, 67 vol% ethanol, 68 vol% ethanol, 69 vol% ethanol or 70 vol% ethanol, etc.

[0048] Preferably, in step (1), the baking time is 15 to 20 minutes, for example, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes or 20 minutes, and the baking temperature is 55 to 65°C, for example, 55°C, 57°C, 60°C, 63°C or 65°C.

[0049] Preferably, in step (1), the soaking time is 5 to 20 min, for example, 5 min, 8 min, 10 min, 12 min, 15 min, 16 min, 18 min or 20 min, and the soaking temperature is 20 to 25°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C.

[0050] Preferably, in step (2), the soaking time is 5 to 20 min, for example, 5 min, 8 min, 10 min, 12 min, 15 min, 16 min, 18 min or 20 min, and the soaking temperature is 20 to 25°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C, etc.

[0051] Preferably, in step (3), the soaking time in anhydrous ethanol, 85-90 vol% ethanol, 65-70 vol% ethanol and purified water is each independently 1-20 min, for example, it can be 1 min, 2 min, 4 min, 6 min, 8 min, 10 min, 12 min, 14 min, 16 min, 18 min or 20 min, and the soaking temperature is each independently 20-25°C, for example, it can be 20°C, 21°C, 22°C, 23°C, 24°C or 25°C, etc.

[0052] In a fourth aspect, the present invention provides a use of the environmentally friendly dewaxing and transparent reagent as described in the first aspect in preparing pathological tissue sections.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) The dewaxing and transparent reagent is miscible with paraffin and ethanol, and makes the slices more uniform and transparent by reducing the tissue refractive index, and the slice structure is clear. The raw materials of the dewaxing and transparent reagent are non-toxic and safe products, which are easy to use and can be used in general laboratories without the need for use in a fume hood.

[0055] (2) The dewaxing transparent reagent is a mixture of normal paraffin and isoparaffin, which can be used for dewaxing at room temperature, and the solution is clear and not turbid, has low volatility, and is easy to use.

[0056] (3) By adding a transparent agent, the tissue sections can be made more transparent, the solution after dissolving the wax can be more stable, and the dissolved paraffin will not precipitate. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a test result diagram of a breast cancer tissue section sample treated with the dewaxing and transparentizing reagent of Example 1 in Test Example 1 (magnification 1000×).

[0058] Figure 2 This is a test result diagram of a lung cancer tissue section sample treated with a dewaxing transparent reagent in Comparative Example 2 of Test Example 1 (magnification 1000×).

[0059] Figure 3 This is a test result diagram of a lung cancer tissue section sample treated with a dewaxing transparent reagent in Comparative Example 4 of Test Example 1 (magnification 1000×).

[0060] Figure 4 This is a diagram showing the volatility verification results of the dewaxing transparent reagent in Test Example 2 (left, the dewaxing transparent reagent described in Example 4; right, Nanchang Rain Dew Waxing Transparent Liquid).

[0061] Figure 5This is a test result diagram of a thyroid tissue section sample treated with the dewaxing and transparentizing reagent of Example 5 in Test Example 3 (magnification 20×).

[0062] Figure 6 This is a test result diagram of a tonsil tissue section sample treated with the dewaxing and transparentizing reagent of Example 5 in Test Example 3 (magnification 10×).

[0063] Figure 7 This is a test result diagram of a tonsil tissue section sample treated with the dewaxing and transparentizing reagent of Example 5 in Test Example 3 (magnification 20×).

[0064] Figure 8 This is a test result diagram of the appendix tissue section sample treated with the dewaxing and transparentizing reagent of Example 5 in Test Example 3 (magnification 10×).

[0065] Fig. 9 This is a test result diagram of the appendix tissue section sample treated with the dewaxing and transparentizing reagent of Example 5 in Test Example 3 (magnification 20×).

[0066] Fig.10 This is a test result diagram of a lung adenocarcinoma tissue section sample treated with the dewaxing and transparentizing reagent of Example 5 in Test Example 3 (magnification 10×).

[0067] Fig.11 This is a test result diagram of a cervical cancer tissue section sample treated with the dewaxing and transparentizing reagent of Example 5 in Test Example 3 (magnification 10×).

[0068] Fig.12 This is a test result diagram of an endometrial tissue section sample treated with the dewaxing and transparentizing reagent of Example 5 in Test Example 3 (magnification 10×). DETAILED DESCRIPTION

[0069] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0070] If no specific techniques or conditions are indicated in the examples, all are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents used or those without manufacturer indication are conventional products that can be purchased through regular channels.

[0071] In the following embodiments, comparative examples and test examples, the sources of various materials are as follows:

[0072]

[0073] Example 1

[0074] This embodiment provides an environmentally friendly dewaxing and transparent reagent, which includes, by weight:

[0075] 1 part of n-hexadecane, 10 parts of isododecane, 1 part of tert-butyl alcohol and 32 # 1.2 parts of white oil.

[0076] The preparation method of the environmentally friendly dewaxing transparent reagent comprises the following steps:

[0077] Take 1 part of n-hexadecane, 10 parts of isododecane, 1 part of tert-butyl alcohol and 32 # 1.2 parts of white oil were stirred electrically at 20° C. for 10 min at a rotation speed of 400 rpm to obtain the environmentally friendly dewaxing transparent reagent.

[0078] Example 2

[0079] This embodiment provides an environmentally friendly dewaxing and transparent reagent, which includes, by weight:

[0080] 1 part of n-dodecane, 10 parts of isohexadecane, 1 part of benzyl benzoate and 32 # 1.2 parts of white oil.

[0081] The preparation method of the environmentally friendly dewaxing transparent reagent comprises the following steps:

[0082] 1 part of n-dodecane, 10 parts of isohexadecane, 1 part of benzyl benzoate and 32 # 1.2 parts of white oil were stirred electrically at 25° C. for 20 min at a rotation speed of 300 rpm to obtain the environmentally friendly dewaxing transparent reagent.

[0083] Example 3

[0084] This embodiment provides an environmentally friendly dewaxing and transparent reagent, which includes, by weight:

[0085] 3 parts of n-decane, 15 parts of isoundecane, 1 part of benzyl benzoate and 32 # 1.2 parts of white oil.

[0086] The preparation method of the environmentally friendly dewaxing transparent reagent comprises the following steps:

[0087] Take 3 parts of n-decane, 15 parts of isoundecane, 1 part of benzyl benzoate and 32 # 1.2 parts of white oil were magnetically stirred at 24° C. for 15 min at a rotation speed of 200 rpm to obtain the environmentally friendly dewaxing transparent reagent.

[0088] Example 4

[0089] This embodiment provides an environmentally friendly dewaxing and transparent reagent, which includes, by weight:

[0090] 3 parts of n-heptane, 12 parts of isohexadecane, 2 parts of benzyl benzoate and 15 # 3 parts white oil.

[0091] The preparation method of the environmentally friendly dewaxing transparent reagent comprises the following steps:

[0092] Take 3 parts of n-heptane, 12 parts of isohexadecane, 2 parts of benzyl benzoate and 15 # 3 parts of white oil were magnetically stirred at 25° C. for 7 min at a rotation speed of 200 rpm to obtain the environmentally friendly dewaxing transparent reagent.

[0093] Example 5

[0094] This embodiment provides an environmentally friendly dewaxing and transparent reagent, which includes, by weight:

[0095] 3 parts of n-decane, 12 parts of isomeric tetradecane, 2 parts of N,N,N,N-tetrakis(2-hydroxypropyl)ethylenediamine and 32 # 1.2 parts of white oil.

[0096] The preparation method of the environmentally friendly dewaxing transparent reagent comprises the following steps:

[0097] Take 3 parts of n-decane, 12 parts of isomeric tetradecane, 2 parts of N,N,N,N-tetrakis(2-hydroxypropyl)ethylenediamine and 32 # 1.2 parts of white oil were stirred electrically at 20° C. for 20 min at a rotation speed of 800 rpm to obtain the environmentally friendly dewaxing transparent reagent.

[0098] Example 6

[0099] This embodiment provides an environmentally friendly dewaxing transparent reagent, which is different from Embodiment 1 only in that the weight portion of n-hexadecane is 0.5 parts.

[0100] The preparation method of the environmentally friendly dewaxing transparent reagent is referred to Example 1, and the dewaxing transparent reagent is prepared and mixed according to the formula amount. The dewaxing transparent reagent cannot dissolve paraffin and is milky white.

[0101] Example 7

[0102] This embodiment provides an environmentally friendly dewaxing transparent reagent, which is different from Embodiment 1 only in that the weight portion of n-hexadecane is 6 parts.

[0103] The preparation method of the environmentally friendly dewaxing transparent reagent refers to Example 1, and the dewaxing transparent reagent is prepared by mixing according to the formula. The dewaxing transparent reagent can dissolve paraffin under heating conditions, but when the temperature is lowered, the paraffin will re-solidify and dewaxing cannot be achieved.

[0104] Example 8

[0105] This embodiment provides an environmentally friendly dewaxing transparent reagent, which is different from Embodiment 1 only in that the weight portion of the isododecane is 1 part.

[0106] The preparation method of the environmentally friendly dewaxing transparent reagent is referred to Example 1, and the dewaxing transparent reagent is prepared by mixing according to the formula amount. The dewaxing transparent reagent cannot dissolve paraffin.

[0107] Example 9

[0108] This embodiment provides an environmentally friendly dewaxing transparent reagent, which is different from Embodiment 1 only in that the weight portion of the isododecane is 20 parts.

[0109] The preparation method of the environmentally friendly dewaxing transparent reagent refers to Example 1, and the dewaxing transparent reagent is prepared by mixing according to the formula. The dewaxing transparent reagent can dissolve paraffin under heating conditions, but when the temperature is lowered, the paraffin will re-solidify and dewaxing cannot be achieved.

[0110] Comparative Example 1

[0111] This comparative example provides an environmentally friendly dewaxing transparent reagent. Compared with Example 1, the environmentally friendly dewaxing transparent reagent does not contain n-hexadecane;

[0112] The preparation method of the environmentally friendly dewaxing transparent reagent is referred to Example 1, and the dewaxing transparent reagent is prepared by mixing according to the formula amount. The dewaxing transparent reagent cannot dissolve paraffin.

[0113] Comparative Example 2

[0114] This comparative example provides an environmentally friendly dewaxing and transparent reagent. Compared with Example 1, the environmentally friendly dewaxing and transparent reagent does not contain tert-butyl alcohol;

[0115] The preparation method of the environmentally friendly dewaxing transparent reagent is shown in Example 1.

[0116] Comparative Example 3

[0117] This comparative example provides an environmentally friendly dewaxing transparent reagent. Compared with Example 1, the environmentally friendly dewaxing transparent reagent does not contain 32 # White oil;

[0118] The preparation method of the environmentally friendly dewaxing transparent reagent is referred to Example 1, and the dewaxing transparent reagent is prepared by mixing according to the formula amount. The dewaxing transparent reagent does not contain 32# White oil: During the pouring process, a large amount of liquid remains along the outer wall of the container, contaminating the desktop and the outer wall of the reagent bottle.

[0119] Comparative Example 4

[0120] This comparative example provides an environmentally friendly dewaxing transparent reagent. Compared with Example 1, the environmentally friendly dewaxing transparent reagent does not contain tert-butyl alcohol and 32 # White oil;

[0121] The preparation method of the environmentally friendly dewaxing transparent reagent is based on Example 1, and the dewaxing transparent reagent is prepared according to the formula amount. The dewaxing transparent reagent does not contain tert-butyl alcohol and 32 # White oil: During the pouring process, a large amount of liquid remains along the outer wall of the container, contaminating the desktop and the outer wall of the reagent bottle.

[0122] Test Example 1

[0123] The environmentally friendly dewaxing and transparent reagents described in Example 1, Comparative Example 2 and Comparative Example 4 were used to perform a fluorescence in situ hybridization experiment, which included the following steps:

[0124] Ten neutral formalin-fixed paraffin-embedded section samples were collected, including breast cancer tissue section samples and lung cancer tissue section samples, and the thickness of the section samples was 3 μm.

[0125] The probe used in the breast cancer tissue section sample is a HER2 probe, and the probe used in the lung cancer tissue section sample is a RET (10q11) gene break probe.

[0126] The slice samples were baked on a hot plate for 20 minutes at a temperature of 60° C. After baking, the slice samples were respectively placed in the environmentally friendly dewaxing transparent reagents described in Example 1 and Comparative Example 2 and soaked in 2 cylinders for 10 minutes. The slice samples were taken out and soaked in anhydrous ethanol at 25° C. for 10 minutes, and then sequentially placed in anhydrous ethanol, 90 vol% ethanol and 70 vol% ethanol for 3 minutes each, and then placed in purified water for 3 minutes, and excess water was shaken off.

[0127] Boil the slices in 99℃ purified water for 25min, remove the slides and air dry at room temperature. Add 200μL of pepsin preheated at 37℃ to the sample area for digestion. Shake off the excess liquid, soak in 2×SSC buffer at room temperature for 3min, then soak in anhydrous ethanol, 90vol% ethanol and 70vol% ethanol for 2min each, remove the slices and air dry at room temperature.

[0128] Take out the probe from the -20℃ refrigerator, shake and mix, centrifuge briefly, add 10μL of probe to the hybridization area, cover with a 22×22mm cover glass, press lightly to evenly distribute the probe, and avoid bubbles. Seal the slide along the edge of the cover glass with rubber cement to completely cover the contact part between the cover glass and the slide.

[0129] Place the slide in the hybridizer, moisten the wet strip of the in situ hybridizer, place the wet strip in place, cover the hybridizer cover, set the "Denat&Hyb" program, denature at 85°C for 5 minutes, and hybridize at 37°C for 10 hours. After the hybridization is completed, tear off the rubber cement, remove the cover glass, soak it in 37°C 2×SSC buffer for 10 minutes, then soak it in 0.1% NP40 / 2×SSC buffer for 5 minutes, soak it in 70vol% ethanol at room temperature for 3 minutes, take out the slide, and dry it in the dark. Add 10μL of DAPI solution to the dry 22×22mm cover glass, invert the sample slide, make the cover glass contact the target area of ​​the slide, invert and press lightly to remove bubbles, and observe the sliced ​​sample under a microscope.

[0130] Among them, the test results of the tissue section samples treated with the environmentally friendly dewaxing and transparent reagent described in Example 1 are:

[0131] Breast cancer tissue biopsy results Figure 1 As shown, from Figure 1 It can be seen that the tissue digestion is complete, the cell nucleus is clearly exposed, the DPAI staining is uniform, and the contrast with the background is clear. Through the specific channel filter, the signal points of HER2 and CEP17 are bright and the edges are clear.

[0132] The test results of the tissue section samples treated with the environmentally friendly dewaxing and transparent reagents described in Comparative Examples 2 and 4 are as follows:

[0133] The test results of the lung cancer tissue section samples treated with the dewaxing transparent reagent in Comparative Example 2 are as follows: Figure 2 As shown. The dewaxing was performed using the formula of Comparative Example 2. The tissue was visible under the microscope, the pepsin digestion result was normal, and the film reader believed that the tissue background was bright white and could not form a clear contrast with the background. The environmentally friendly dewaxing and transparent reagent described in Comparative Example 2 does not contain tert-butyl alcohol. After the wax is dissolved, the wax will precipitate on the slice sample, the dewaxing effect is poor, the hybridization efficiency is low in the subsequent hybridization experiment, and the signal point is thin, which is not conducive to the observation of FISH results.

[0134] The environmentally friendly dewaxing transparent reagent described in Comparative Example 4 does not contain tert-butyl alcohol and 32 # The test results of lung cancer tissue samples after white oil treatment are as follows Figure 3 The environmentally friendly dewaxing and transparent reagent has a poor dewaxing effect, and the hybridization efficiency is low in the subsequent hybridization experiment, and the signal points are thin, which affects the observation of FISH results.

[0135] Test Example 2

[0136] The volatility of the environmentally friendly dewaxing transparent reagent described in Example 4 was verified, wherein the control was the Nanchang rain dew wax dewaxing transparent liquid.

[0137] Place 50 mg of paraffin wax rolls of slices in a culture dish, the thickness of the wax rolls is 5 μm, and add 10 mL of dewaxing agent. The control is Nanchang rain dew immersion dewaxing transparent liquid.

[0138] Both dewaxing reagents can dissolve wax when left at room temperature for 1 min.

[0139] After testing, after standing at room temperature for 4 days, a white waxy substance appeared in the control, indicating that the solution had basically evaporated. The test results are as follows: Figure 4 After being placed at room temperature for 4 days, the environmentally friendly dewaxing transparent reagent described in Example 4 is still in liquid state, and the Nanchang rain dew wax-immersion dewaxing transparent liquid is a soft waxy mixture.

[0140] Test Example 3

[0141] The environmentally friendly dewaxing and transparent reagent described in Example 5 was used to perform an immunohistochemical experiment, which included the following steps:

[0142] Ten neutral formalin-fixed paraffin-embedded section samples were obtained, including thyroid tissue section samples, tonsil tissue section samples, appendix tissue section samples, lung adenocarcinoma tissue section samples, cervical cancer tissue section samples and endometrial tissue section samples, and the thickness of the section samples was 3 μm.

[0143] The slice samples were baked on a hot plate for 20 minutes at a temperature of 60° C. After baking, the slice samples were respectively placed in the environmentally friendly dewaxing and transparent reagents described in Example 5, Comparative Example 1, Comparative Example 3 and Comparative Example 4 and immersed in 2 cylinders for 10 minutes. The slice samples were taken out and immersed in anhydrous ethanol at 25° C. for 10 minutes, and then immersed in anhydrous ethanol, 90 vol% ethanol and 70 vol% ethanol in turn for 5 minutes each, and immersed in purified water for 5 minutes, and excess water was shaken off.

[0144] Use EDTA solution to fix under high pressure for 2 minutes, take out the sliced ​​sample and shake dry. Use an immunohistochemistry pen to draw a circle 2mm outside the edge of the tissue, then soak it in TPBS for later use, and pay attention to keep the tissue moist. Prepare a fresh 3vol% H2O2 solution with distilled water, shake off the excess water of the sliced ​​sample, add the 3vol% H2O2 solution to the tissue, seal it at room temperature for 10 minutes, rinse it with purified water for 2 minutes, and put it in PBS buffer for later use.

[0145] Remove the slice sample from the PBS buffer and spin dry. After the circle appears, add the ready-to-use primary antibody to the circle and incubate at room temperature for 30 minutes. After the end, rinse off the excess primary antibody reagent with PBS buffer and put it in PBS buffer for use. Remove the slice sample from the PBS buffer and spin dry. After the circle appears, add the secondary antibody to the circle and incubate at room temperature for 20 minutes. After the end, rinse off the excess secondary antibody reagent with PBS buffer and put it in PBS buffer for use. Prepare fresh DAB colorimetric solution, spin dry the slice sample, add DAB colorimetric solution to the circle, incubate for 3 minutes, terminate the staining with purified water, and rinse with tap water 4 times.

[0146] The primary antibody in this test example comes from Guangzhou Anping Pharmaceutical Technology Co., Ltd., and the DAB colorimetric solution comes from Guangzhou Anping Pharmaceutical Technology Co., Ltd., with the product number IBF10-01; the types of primary antibodies and secondary antibodies used for different slice samples in this test example are shown in Table 1.

[0147] Table 1

[0148]

[0149]

[0150] After the slice samples were dried, they were placed in a hematoxylin staining solution tank and soaked for 3 minutes. The slice rack was taken out and rinsed with tap water several times until the excess hematoxylin dye was washed away. After the water was removed, they were placed in PBS buffer solution to return to blue for 1 minute, then rinsed with tap water 5 times, dehydrated and transparently sealed, and the slice samples were observed under a microscope.

[0151] Among them, the test results of the tissue section samples treated with the environmentally friendly dewaxing and transparent reagent described in Example 5 are:

[0152] The results of the thyroid tissue biopsy samples are as follows Figure 5 The test results of tonsil tissue samples are shown in Figure 6 and Figure 7 As shown in Figure 2, the test results of the appendix tissue sample are as follows: Figure 8 and Fig. 9 As shown in Figure 2, the test results of lung adenocarcinoma tissue samples are as follows: Fig.10 As shown in Figure 2, the test results of cervical cancer tissue samples are as follows: Fig.11 As shown, the test results of endometrial tissue sections are as follows Fig.12 As shown in the figure, it can be seen that the slice sample is stained evenly, dark signal expression can be seen in the positive expression area, and there are no obvious droplets in the cell nucleus under a high-power microscope.

[0153] Test Example 4

[0154] The wax dissolving rate and saturated dewaxing rate of the environmentally friendly dewaxing transparent reagent described in Examples 1 to 5 were tested at room temperature.

[0155] Method for determining the wax dissolution rate:

[0156] Pathological section paraffin (Shanghai Huayong Paraffin Co., Ltd.) was used to prepare blank wax blocks, which were sliced ​​at a thickness of 5 μm. A certain weight of the wax roll was weighed, and a certain amount of dewaxing agent was added to dissolve the paraffin, and the time required for dissolution was recorded.

[0157] Determination method of saturated dewaxing rate:

[0158] Pathological section paraffin (Shanghai Huayong Paraffin Co., Ltd.) was prepared into blank wax blocks, sliced ​​at a thickness of 5 μm, a certain weight of the wax roll was weighed, a certain amount of dewaxing agent was added to dissolve the paraffin, and the amount of dewaxing agent used to completely dissolve the paraffin in 20 min was recorded.

[0159] The results of the wax dissolving rate and saturated dewaxing rate of the environmentally friendly dewaxing transparent reagent described in Examples 1 to 5 are shown in Table 2:

[0160] Table 2

[0161] sample Wax dissolving rate Saturated dewaxing rate (after conversion) Example 1 10mg / mL*(4min 58s) 51mg / mL Example 2 10mg / mL*(4min 51s) 47 mg / mL Example 3 20mg / mL*(9min 08s) 52mg / mL Example 4 20mg / mL*(10min 02s) 48mg / mL Example 5 50mg / mL*(21min 04s) 50mg / mL

[0162] As shown in Table 1, it takes 4 min 58 s to dissolve 100 mg of paraffin using 10 mL of the dewaxing transparent reagent in Example 1, 4 min 51 s to dissolve 100 mg of paraffin using 10 mL of the dewaxing transparent reagent in Example 2, 9 min 08 s to dissolve 200 mg of paraffin using 10 mL of the dewaxing transparent reagent in Example 3, 10 min 02 s to dissolve 200 mg of paraffin using 10 mL of the dewaxing transparent reagent in Example 4, and 21 min 04 s to dissolve 500 mg of paraffin using 10 mL of the dewaxing transparent reagent in Example 5. The test results show that the dewaxing transparent reagents in Examples 1 to 5 have a fast wax dissolving rate, can be dewaxed at room temperature, and the solution is clear and not turbid, has low volatility, is easy to use, and is suitable for clinical trials.

[0163] The saturated wax dissolving amount of the environmentally friendly dewaxing and transparent reagent described in Examples 1 to 5 is between 47 and 52 mg / mL. The environmentally friendly dewaxing and transparent reagent has a fast wax dissolving rate and a significant dewaxing and transparent effect.

[0164] In summary, the present invention provides an environmentally friendly dewaxing transparent reagent, which is used in the film making process, has stable wax dissolving, fast wax dissolving speed, low volatility, low odor, and high ignition point, and can reduce the tissue refractive index, making the slices more uniform and transparent, and the slice structure clear. The raw materials of the dewaxing transparent reagent are non-toxic and safe products, with fast wax dissolving rate, high wax dissolving efficiency, and safe and convenient use.

[0165] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An environmentally friendly dewaxing and transparent reagent, characterized in that: The environmentally friendly dewaxing and transparent reagent is a combination of 1 to 2 parts of an initiator, 7 to 10 parts of a wax dissolving agent, 1 to 2 parts of a transparent agent, and 0.5 to 2 parts of a solution modifier; The transparent agent includes any one of tert-butyl alcohol and benzyl benzoate; The initiator comprises any one of n-heptane, n-octane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane or n-hexadecane, or a combination of at least two thereof; The solution improver comprises 7 # ~100 # White oil; The wax dissolving agent includes any one of isomeric decane, isomeric undecane, isomeric dodecane, isomeric tridecane, isomeric tetradecane, isomeric pentadecane or isomeric hexadecane, or a combination of at least two thereof.

2. A method for preparing an environmentally friendly dewaxing transparent reagent according to claim 1, characterized in that: The preparation method comprises the following steps: mixing 1-2 parts of an initiator, 7-10 parts of a wax dissolving agent, 1-2 parts of a transparent agent and 0.5-2 parts of a solution improving agent by weight to obtain the environmentally friendly dewaxing transparent agent.

3. The method for preparing the environmentally friendly dewaxing transparent reagent according to claim 2, characterized in that: The mixing temperature is 20-25°C.

4. The method for preparing the environmentally friendly dewaxing transparent reagent according to claim 2, characterized in that: The mixing method includes electric stirring and / or magnetic stirring.

5. The method for preparing the environmentally friendly dewaxing transparent reagent according to claim 4, characterized in that: The rotation speed of the electric stirring is 200 to 1000 rpm, and the time of the electric stirring is 1 to 30 minutes.

6. The method for preparing the environmentally friendly dewaxing transparent reagent according to claim 4, characterized in that: The rotation speed of the magnetic stirring is 100 to 500 rpm, and the time of the magnetic stirring is 3 to 100 minutes.

7. The method for preparing the environmentally friendly dewaxing transparent reagent according to claim 2, characterized in that: The preparation method comprises the following steps: 1 to 2 parts of an initiator, 7 to 10 parts of a wax dissolving agent, 1 to 2 parts of a transparent agent and 0.5 to 2 parts of a solution modifier are stirred electrically at 20 to 25° C. for 1 to 30 minutes at a speed of 200 to 1000 rpm to obtain the environmentally friendly dewaxing transparent agent; Alternatively, 1 to 2 parts of initiator, 7 to 10 parts of wax dissolving agent, 1 to 2 parts of transparent agent and 0.5 to 2 parts of solution modifier are magnetically stirred at 20 to 25° C. for 3 to 100 minutes at a rotation speed of 100 to 500 rpm to obtain the environmentally friendly dewaxing transparent agent.

8. A method for using the environmentally friendly dewaxing transparent reagent according to claim 1, characterized in that: The method of use comprises the following steps: (1) taking a paraffin-embedded section sample, baking the section and then immersing the section in the environmentally friendly dewaxing and transparent reagent; (2) soaking the slice sample soaked in step (1) in anhydrous ethanol; (3) The sliced ​​samples soaked in step (2) are sequentially soaked in anhydrous ethanol, 85-90 vol% ethanol, 65-70 vol% ethanol and purified water.

9. The method for using the environmentally friendly dewaxing transparent reagent according to claim 8, characterized in that: In step (1), the baking time is 15 to 20 minutes, and the baking temperature is 55 to 65°C.

10. The method for using the environmentally friendly dewaxing transparent reagent according to claim 8, characterized in that: In step (1), the soaking time is 5 to 20 minutes, and the soaking temperature is 20 to 25°C.

11. The method for using the environmentally friendly dewaxing transparent reagent according to claim 8, characterized in that: In step (2), the soaking time is 5 to 20 minutes, and the soaking temperature is 20 to 25°C.

12. The method for using the environmentally friendly dewaxing transparent reagent according to claim 8, characterized in that: In step (3), the soaking time in anhydrous ethanol, 85-90 vol% ethanol, 65-70 vol% ethanol and purified water is independently 1-20 min, and the soaking temperature is independently 20-25°C.

13. Use of the environmentally friendly dewaxing and transparent reagent according to claim 1 in preparing pathological tissue sections.

Images