A red stain, reagent combination and method for masson's trichrome staining

Abstract

The application discloses a red dye solution, a reagent combination and a method for Masson trichrome staining, and belongs to the field of histochemistry technology. The red dye solution comprises 0.4-1.0% of bibracine, 0.4-1.0% of temptation red and 1-2% of acetic acid, and water is used as a solvent, and the red dye solution further comprises 0.05-0.15% of orange G sodium salt; the reagent combination comprises the red dye solution and further comprises a blue dye solution, the blue dye solution comprises 2% of aniline blue, 1-2% of acetic acid, and water is used as a solvent, and the blue dye solution further comprises 0.005-0.03% of fast green, 0.0025-0.015% of brilliant green or 0.005-0.03% of bromocresol green sodium salt. The reagent combination further comprises hematoxylin dye solution, red differentiation agent and blue differentiation agent. The improved red dye solution for Masson trichrome staining provided by the application increases the affinity for muscle fiber dyeing, makes the muscle fiber dyeing more firm, and can still maintain bright red after phosphomolybdic acid differentiation. Meanwhile, the improved blue dye solution makes the collagen fiber dyeing more bright, and the color contrast between the muscle fiber and the collagen fiber is more obvious and easier to distinguish.

Description

Technical Field

[0001] This invention relates to the field of histochemistry, and more specifically, to a red staining solution, reagent combination, and method for Masson's trichrome staining. Background Technology

[0002] Masson staining is commonly used for the differential staining of collagen fibers and muscle fibers, and is one of the important methods for preparing pathological tissue slides. Collagen fibers can appear in the body during chronic inflammation, organization, and scar formation, as the pathological process progresses. In early HE-stained sections, these fibers are often difficult to distinguish from fibrin, but Masson staining can confirm their presence.

[0003] Currently, the Masson staining method mainly uses Ponceau S and Acid Fuchsin to stain muscle fibers, and can further add Bressy Scarlet; aniline blue is used to stain collagen fibers. For example, Chinese invention patent CN110926911A discloses a Masson staining kit and its staining method. The Masson staining kit includes Ponceau S, Acid Fuchsin, and Aniline Blue staining solutions. The Ponceau S, Acid Fuchsin staining solution is prepared by dissolving 4g of Ponceau S and 2g of Acid Fuchsin in 495mL of distilled water, and then adding 5mL of glacial acetic acid. The Aniline Blue staining solution is prepared by dissolving 10g of Aniline Blue in 490mL of distilled water to obtain solution B, and then adding 10mL of glacial acetic acid to solution B. For example, Xu Weiyong et al. disclosed a modified Masson trichrome staining method, with the following staining solution: 30 mL of 1% Ponceau 2R aqueous solution, 20 mL of 1% acid fuchsin solution, 50 mL of 1% Bibruce Scarlet aqueous solution, and 1 mL of glacial acetic acid (Xu Weiyong, Yang Qun, Fan Xiaoli; Introducing a modified Masson trichrome staining method [J]; Journal of Diagnostic Pathology; 2005, No. 1).

[0004] However, because Ponceau S and Acid Fuchsin do not stain muscle fibers well enough, they are easily destained through differentiation or washing. As a result, conventional Masson trichrome staining produces darker and lighter red stains on muscle fibers and cytoplasm, and the color contrast of tissue structures is not bright and clear enough or difficult to distinguish. After counterstaining with blue, the red is easily covered up, becoming darker, bluish-purple, or even indistinguishable. Summary of the Invention

[0005] To address at least one of the aforementioned technical problems, the present invention aims to provide a Masson trichrome staining solution that produces brighter and more durable staining, with clear contrast and easy identification of tissue structure colors. To achieve this objective, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a red dye solution for Masson's trichrome staining, comprising 0.4% to 1.0% by mass / volume of Bibrese Scarlet, 0.4% to 1.0% by mass / volume of Allura Red, and 1% to 2% by volume of acetic acid, with water as the solvent.

[0007] In this invention, Bibrese Scarlet and Allura Red are used instead of Ponceau S and Acid Fuchsin. Bibrese Scarlet and Allura Red are azo dyes with a molecular weight of 400-600. Their staining effect on muscle fibers is more stable than that of Ponceau S and Acid Fuchsin, making the red color of the tissue more vivid and the contrast with blue more distinct.

[0008] In some embodiments of the present invention, the red dye solution further includes 0.05% to 0.15% by mass / volume of orange-yellow G sodium salt. The orange-yellow G sodium salt is an orange-yellow azo dye with a molecular weight of 400-600, which can adjust the red dye to prevent excessive staining of muscle fibers. Using orange-yellow G sodium salt to adjust the red color prevents the red and blue dye solutions from overlapping and staining the tissue too deeply or too darkly, making the red staining of muscle fibers and the blue staining of collagen fibers more distinct.

[0009] A second aspect of the present invention provides a reagent combination for Masson's trichrome staining, comprising any of the red staining solutions described in the first aspect of the present invention, and further comprising a blue staining solution, wherein the blue staining solution comprises 2% aniline blue and 1% to 2% acetic acid by volume, and the solvent is water.

[0010] In some embodiments of the present invention, the blue dye solution further includes 0.005% to 0.03% by weight of Fast Green, 0.0025% to 0.015% by weight of Brilliant Green, or 0.005% to 0.03% by weight of bromocresol green sodium salt. Using Fast Green, Brilliant Green, or bromocresol green sodium salt to adjust the aniline blue dye solution can prevent the blue dye solution from staining the collagen fibers too darkly.

[0011] Furthermore, the reagent combination also includes hematoxylin staining solution, red differentiating agent and / or blue differentiating agent.

[0012] In some embodiments of the present invention, the hematoxylin staining solution is Weigert iron hematoxylin staining solution, which is prepared by mixing Weigert iron hematoxylin A solution and Weigert iron hematoxylin B solution in a ratio of 1:2 to 2:1.

[0013] The Weigert iron hematoxylin A solution contains 0.5% to 2.0% hematoxylin by mass and volume, 10% to 20% ethylene glycol by volume, and 95% ethanol as the solvent.

[0014] The Weigert iron hematoxylin B solution contains 0.36% to 0.38% HCl by mass, 1.0% to 1.5% FeCl3 by mass and volume, and water as the solvent.

[0015] In some specific embodiments of the present invention

[0016] The preparation method for 50mL Weigert iron hematoxylin A solution is as follows: weigh 0.5g of hematoxylin, add 7mL of ethylene glycol, then add 43mL of 95% ethanol, and mix well.

[0017] The preparation method for 50 mL Weigert's iron hematoxylin B solution is as follows: Dissolve 0.5 mL of concentrated hydrochloric acid in 50 mL of purified water, add 0.58 g of anhydrous ferric chloride, and mix well. The concentrated hydrochloric acid has a mass fraction of 36%–38%.

[0018] In some preferred embodiments of the present invention, the Weigert iron hematoxylin staining solution is prepared by mixing Weigert iron hematoxylin A solution and Weigert iron hematoxylin B solution in a 1:1 ratio.

[0019] In some embodiments of the present invention, the red differentiating agent is a phosphotungstic phosphotomolybdic acid solution, which includes 2.0% to 3.0% by mass and volume of phosphotungstic acid and phosphotomolybdic acid by mass and volume, and the solvent is water.

[0020] In some specific embodiments of the present invention, the method for preparing 100 mL of the phosphotungstic phosphomolybdic acid solution is as follows: weigh 2.5 g of phosphotungstic acid and 2.5 g of phosphomolybdic acid, and add 100 mL of water to dissolve them.

[0021] In some embodiments of the present invention, the blue differentiating agent is an acetic acid solution, which includes 0.5% to 2% acetic acid by volume, and the solvent is water.

[0022] In some specific embodiments of the present invention, the 500 mL acetic acid solution is prepared by adding 5 mL of acetic acid to 500 mL of purified water and mixing well.

[0023] The third aspect of the present invention provides the use of a reagent combination for Masson's trichrome staining as described in any of the second aspects of the present invention in the preparation of a kit for tissue trichrome staining.

[0024] The trichrome staining refers to staining cell nuclei and selectively displaying collagen fibers and muscle fibers. The trichrome staining process is related to the size of the anionic dye molecules and tissue penetration: molecular size is reflected in molecular weight; small molecular weights easily penetrate dense, low-permeability tissues, while large molecular weights can only enter loosely structured, high-permeability tissues. However, aniline blue has a very large molecular weight; therefore, after trichrome staining, muscle fibers and cytoplasm appear red, collagen fibers appear blue, and cell nuclei appear purplish-brown or bluish-brown. The trichrome staining method is mainly used to distinguish collagen fibers and muscle fibers, making tissue structures easier to identify.

[0025] In this invention, the tissue refers to tissue comprising collagen fibers and / or muscle fibers. Using the improved Masson's trichrome staining solution described above for staining collagen fibers and muscle fibers allows for the analysis and assessment of fibrous tissue morphology and fibrotic lesions in organs such as the heart, kidneys, and liver.

[0026] In some embodiments of the present invention, the tissue is selected from at least one of the group consisting of cervical tissue, colon tissue, kidney tissue, and liver tissue.

[0027] A third aspect of the present invention provides a method for trichrome staining of tissues using a reagent combination comprising red staining solution, blue staining solution, hematoxylin staining solution, red differentiating agent, and blue differentiating agent, as described in the second aspect of the present invention, comprising the following steps:

[0028] S1, heat and bake the slides containing tissue samples, then dewax and hydrate them;

[0029] S2, stain the tissue with hematoxylin solution for 5-10 minutes, then wash away excess hematoxylin solution;

[0030] S3, incubate and stain with the red dye solution for 5-10 minutes, then wash away excess red dye solution;

[0031] S4, use the red differentiating agent to differentiate for 1-3 minutes, then wash away excess red differentiating agent;

[0032] S5, incubate and stain with the blue dye solution for 5-10 minutes, then wash away excess blue dye solution;

[0033] S6, differentiate using the blue differentiating agent for 0.5 to 2 minutes, then wash away excess blue differentiating agent;

[0034] S7, after dehydration and clearing, was dried and sectioned, and then mounted with neutral resin. The staining was observed under a microscope.

[0035] The hematoxylin staining solution is Weigert iron hematoxylin staining solution, which is prepared by mixing Weigert iron hematoxylin A solution and Weigert iron hematoxylin B solution in a ratio of 1:2 to 2:1 before use.

[0036] Beneficial effects of the present invention

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The red staining solution for Masson's trichrome staining of this invention uses Brice Scarlet and Allura Red instead of Ponceau S and Acid Fuchsin, resulting in a more vivid contrast between the red and blue staining of the tissue. Furthermore, the red staining solution is adjusted with Sodium Orange G, effectively preventing the red and blue staining solutions from overlapping and staining the tissue too deeply or too darkly. Even further, the blue staining solution in the reagent combination for Masson's trichrome staining of this invention uses Saxo Green, Brilliant Green, or Sodium Bromocresol Green to adjust the aniline blue staining solution, effectively preventing the blue staining solution from staining collagen fibers too deeply.

[0039] The improved red dye solution for Masson's trichrome staining provided by this invention increases the affinity for staining muscle fibers, making the staining more robust and maintaining a vibrant red color even after phosphomolybdic acid differentiation. Simultaneously, the improved blue dye solution provides more vivid staining of collagen fibers, making the hue contrast between muscle fibers and collagen fibers more pronounced and easier to distinguish. Attached Figure Description

[0040] Figure 1 The diagram shows different staining effects on liver tissue in an embodiment of the present invention. In the diagram, A: staining result marked "0"; B: staining result marked "-1"; C: staining result marked "+1"; D: staining result marked "+2"; E: staining result marked "+3".

[0041] Figure 2 The diagram illustrates different staining effects on kidney tissue in embodiments of the present invention. In the diagram, A: staining result marked "0"; B: staining result marked "-1"; C: staining result marked "+1"; D: staining result marked "+2"; E: staining result marked "+3". Detailed Implementation

[0042] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0043] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. The numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided that there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed lowest and highest values ​​are considered to be clearly described in this application.

[0044] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0045] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0046] Example

[0047] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0049] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0051] Example 1: Formulation optimization of Masson's red dye solution

[0052] 1. Preparation of red and blue dye solutions

[0053] This embodiment provides 16 Masson red staining formulations A0-A15, and also provides 2 control red staining formulations, as shown in Table 1:

[0054] Table 1 Formula for 100mL Red Dye Solution

[0055]

[0056]

[0057] Among them, RA1 adopts the formula disclosed in Chinese invention patent CN110926911A; RA2 adopts the formula in the reference (Xu Weiyong, Yang Qun, Fan Xiaoli; Introducing a modified Masson trichrome staining method [J]; Journal of Diagnostic Pathology; 2005 No.1); A0 is a formula that uses Bibrese scarlet instead of Ponceau S in RA2.

[0058] The blue dye solution uses the formula disclosed in Chinese invention patent CN110926911A, numbered RB1.

[0059] RB1: Weigh 2g of aniline blue and 2mL of acetic acid, and add water to 100mL.

[0060] Other reagents and their corresponding preparation methods are as follows:

[0061] Weigert Iron Hematoxylin A Solution: Weigh 0.5g of hematoxylin, add 7mL of ethylene glycol, then add 43mL of 95% ethanol, mix well and set aside.

[0062] Weigert's iron hematoxylin B solution: Dissolve 0.5 mL of concentrated hydrochloric acid in 50 mL of purified water, add 0.58 g of anhydrous ferric chloride, mix well and set aside.

[0063] Weigert iron hematoxylin A solution and B solution are mixed in equal proportions before use to obtain Weigert iron hematoxylin staining solution.

[0064] Phosphotungstic acid-phosphomolybdic acid solution (red differentiating agent): Weigh 2.5g phossphotungstic acid and 2.5g phosphomolybdic acid, add 100mL purified water, and dissolve for later use.

[0065] Acetic acid solution (blue differentiating agent): Add 5 mL of acetic acid to 500 mL of purified water, mix well and set aside.

[0066] 2. Test Organization

[0067] Each red staining solution (RA1, RA2, R0-R15) was combined with RB1 to form staining solutions for testing. Each staining solution combination was used to test four types of tissues: cervix, colon, kidney, and liver. The sample volume for each reagent on each slide was 0.2 mL (preferably enough to completely cover the tissue).

[0068] 3. Testing Methods

[0069] (1) The slides containing tissue samples are heated and baked, and then dewaxed and hydrated.

[0070] (2) Stain with Weigert iron hematoxylin solution for 8 minutes, then rinse with tap water to remove excess Weigert iron hematoxylin solution.

[0071] (3) Incubate with red dye for 8 minutes, then rinse with tap water to remove excess red dye.

[0072] (4) Use red differentiating agent (phosphotungstic phosphotomolybdic acid solution) to differentiate for 2 minutes, then rinse with tap water to remove excess red differentiating agent.

[0073] (5) Incubate with blue dye for 8 minutes, then rinse with tap water to remove excess blue dye.

[0074] (6) Use blue differentiating agent (acetic acid solution) to differentiate for 1 minute, then rinse with tap water to remove excess blue differentiating agent.

[0075] Finally, after dehydration and clearing, the sections were dried, mounted with neutral resin, and the staining was observed under a microscope.

[0076] 4. Test Results

[0077] The test results are shown in Table 2:

[0078] Table 2. Staining results of each red dye combined with RB1

[0079] Red dye number cervix colon kidney liver Overall score RA1 0 0 0 0 0 RA2 +1 0 +1 0 +2 A0 0 0 -1 -1 -2 A1 0 0 0 0 0 A2 +1 +1 0 +1 +3 A3 +1 +1 +1 +1 +4 A4 +1 +1 +1 +1 +4 A5 +1 0 +1 +1 +3 A6 +1 +1 +1 +1 +4 A7 +1 +1 +1 +1 +4 A8 0 0 0 +1 +1 A9 +1 +1 +1 +1 +4 A10 +2 +2 +2 +2 +8 A11 +2 +2 +2 +2 +8 A12 +2 +2 +1 +2 +7 A13 +2 +2 +2 +2 +8 A14 +1 +1 +1 +1 +4 A15 +2 +2 +2 +2 +8

[0080] Result determination:

[0081] Collagen fibers appear blue, muscle fibers and cytoplasm appear red, cell nuclei appear purplish-brown or bluish-brown, and erythrocytes appear orange-red. Using the traditional formula (RA1+RB1) as a baseline, a value of 0 is assigned. Optimization is indicated by a "+", and deterioration by a "-", with numbers representing the degree of optimization / deterioration: +1, +2, ..., -1, -2, ... Optimization refers to brighter staining colors, stronger contrast, and clearer differentiation of tissue and cell structures, making them easier to distinguish. Deterioration indicates excessively light or dark staining, decreased contrast, and insufficient differentiation of tissue and cell structures, making them difficult to distinguish. For liver tissue, the staining results labeled 0, +1, +2, +3, and -1 are as follows... Figure 1 As shown; for kidney tissue, the staining results labeled 0, +1, +2, +3, -1 are as follows. Figure 2 As shown.

[0082] Table 2 shows that RA2, compared to RA1, did not improve staining performance in colon and liver tissues (0), but improved staining performance in cervical and kidney tissues (+1), indicating that adding Bibrese Scarlet to Ponceau S and Acid Fuchsin can improve staining performance, but the improvement is not significant. Further replacement of Ponceau S with Bibrese Scarlet (A0) did not improve staining performance in cervical and colon tissues (0), and even worsened staining performance in kidney and liver tissues (-1).

[0083] Replacing Ponceau S and Acid Fuchsin with Bibruce Scarlet and Allura Red at different addition ratios resulted in varying degrees of improvement in staining outcomes for different tissues. Muscle fibers showed vibrant red staining, collagen fibers showed bright blue staining, and the contrast between muscle and collagen fibers was clear and easily distinguishable, making tissue and cell structures clearly discernible—significantly superior to the previous staining results. However, if the ratio of Bibruce Scarlet to Allura Red was greater than 2.5 times, or the ratio of Allura Red to Bibruce Scarlet was greater than 2.5 times, the staining effect showed almost no improvement (e.g., A1 and A8).

[0084] Based on Bressy Scarlet and Allura Red, the inventors further added Sodium Orange G for testing. They found that the addition of Sodium Orange G further optimized the staining effect. When the amount of Sodium Orange G added was 0.05–0.15 g, the staining effect was significantly improved, as shown in A10–A13 (staining results marked as +2). When A9 and A14 were combined with RB1 separately, the improvement in staining effect was not ideal (staining results marked as +1), indicating that too much or too little Sodium Orange G added would affect the staining effect. The staining result of A15 shows that reducing the amount of acetic acid had no effect on the staining result, indicating that the preferred red dye solution of this embodiment can reduce the amount of acetic acid used.

[0085] Example 2: Preparation of Blue Dye Solution

[0086] To further improve the staining effect, the inventors optimized the blue staining process. To this end, the inventors provided 15 Masson blue dye formulations B1-B15, using RB1 as a control. The formulations of each blue dye are shown in Table 3.

[0087] Table 3 Formula for 100mL Blue Dye Solution

[0088]

[0089]

[0090] Each blue dye solution was tested in combination with A11 from Example 1, using the same tissue and method as in Example 1. The staining results are shown in Table 4.

[0091] Table 4. Staining results of each blue dye combined with A11

[0092] Blue dye number cervix colon kidney liver Overall score RB1 +2 +2 +2 +2 +8 B1 +3 +2 +2 +3 +10 B2 +3 +3 +3 +3 +12 B3 +3 +3 +3 +3 +12 B4 +3 +3 +2 +3 +11 B5 +3 +2 +3 +2 +10 B6 +3 +2 +3 +2 +10 B7 +3 +3 +3 +3 +12 B8 +3 +3 +2 +3 +11 B9 +3 +2 +2 +3 +10 B10 +3 +3 +3 +3 +12 B11 +3 +3 +2 +3 +11 B12 +3 +2 +2 +3 +10 B13 +3 +3 +3 +3 +12 B14 +3 +3 +3 +3 +12 B15 +3 +3 +3 +3 +12

[0093] As shown in Table 4, adding appropriate amounts of Fast Green FCF (B1-B4), Brilliant Green (B5-B8), or bromocresol green sodium salt (B9-B12) to aniline blue can further improve the staining effect to varying degrees, with optimization for different tissues even reaching +3. The staining results from B13-B15 show that reducing the amount of acetic acid had no effect on the staining results, indicating that the preferred blue staining solution in this embodiment can reduce the amount of acetic acid used.

[0094] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A red dye solution for Masson's trichrome staining, characterized in that, It includes 0.4%–1.0% by mass and volume of Bibrese Scarlet, 0.4%–1.0% by mass and volume of Allura Red, and 1%–2% by volume of acetic acid, with water as the solvent.

2. The red dye solution for Masson's trichrome staining according to claim 1, characterized in that, It also includes orange-yellow G sodium salt with a mass-volume fraction of 0.05% to 0.15%.

3. A reagent combination for Masson's trichrome staining, characterized in that, The solution includes the red dye solution as described in claim 1 or 2, and also includes a blue dye solution, wherein the blue dye solution comprises 2% aniline blue and 1% to 2% acetic acid by volume, and the solvent is water.

4. The reagent combination for Masson's trichrome staining according to claim 3, characterized in that, The blue dye solution also includes 0.005% to 0.03% by mass and volume of Fast Green, 0.0025% to 0.015% by mass and volume of Brilliant Green, or 0.005% to 0.03% by mass and volume of bromocresol green sodium salt.

5. The reagent combination for Masson's trichrome staining according to claim 4, characterized in that, It also includes hematoxylin staining solution, red differentiating agent and / or blue differentiating agent.

6. The reagent combination for Masson's trichrome staining according to claim 5, characterized in that, The hematoxylin staining solution is Weigert iron hematoxylin staining solution, which is prepared by mixing Weigert iron hematoxylin A solution and Weigert iron hematoxylin B solution in a ratio of 1:2 to 2:

1. The Weigert iron hematoxylin A solution contains 0.5% to 2.0% hematoxylin by mass and volume, 10% to 20% ethylene glycol by volume, and 95% ethanol as the solvent. The Weigert iron hematoxylin B solution contains 0.36%–0.38% HCl by mass, 1.0%–1.5% FeCl3 by mass (volume fraction), and water as the solvent. The red differentiating agent is a phosphotungstic phosphomolybdic acid solution, which comprises 2.0%–3.0% phosphotungstic acid and phosphomolybdic acid by mass and volume, respectively, and water is used as the solvent. The blue differentiating agent is an acetic acid solution, which contains 0.5% to 2% acetic acid by volume, and the solvent is water.

7. The use of the reagent combination for Masson's trichrome staining as described in any one of claims 3 to 6 in the preparation of a kit for tissue trichrome staining.

8. The application according to claim 7, characterized in that, The tissue is a tissue comprising collagen fibers and / or muscle fibers.

9. The application according to claim 8, characterized in that, The tissue is selected from at least one of the group consisting of cervical tissue, colon tissue, kidney tissue, and liver tissue.

10. A method for performing trichrome staining on tissues using the reagent combination for Masson's trichrome staining as described in claim 6, characterized in that, Includes the following steps: S1, heat and bake the slides containing tissue samples, then dewax and hydrate them; S2, use the hematoxylin staining solution to stain the tissue for 5-10 minutes, then wash away the excess hematoxylin staining solution; S3, incubate and stain with the red dye solution for 5-10 minutes, then wash away excess red dye solution; S4, use the red differentiating agent to differentiate for 1-3 minutes, then wash away excess red differentiating agent; S5, incubate and stain with the blue dye solution for 5-10 minutes, then wash away excess blue dye solution; S6, use the blue differentiating agent to differentiate for 0.5 to 2 minutes, then wash away excess blue differentiating agent; S7, after dehydration and clearing, was dried and sectioned, and then mounted with neutral resin. The staining was observed under a microscope.

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