Multicolor composite dyeing kit, dyeing method and application thereof

Abstract

The present invention relates to the technical field of tissue section staining, and in particular to a multicolor composite staining kit, a staining method, and applications thereof. The multicolor composite staining kit provided by the present invention not only achieves good staining effects and has a high degree of differentiation in staining different tissue structures, but also does not contain hazardous chemical components such as picric acid, ethanol, and ammonia, greatly reducing the associated risks and costs during production, storage, transportation, use, and disposal. In addition, the use of the kit of the present invention can effectively simplify the operating steps and reduce the difficulty of operation. While significantly improving the repeatability of the staining results, it is more environmentally and operator-friendly, has lower costs, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue section staining, in particular to a multi-color composite staining kit, a staining method and applications thereof. Background Art

[0002] Connective tissue, in a narrow sense, refers to the three types of fibers it contains: collagen, reticular fibers, and elastic fibers. In H&E staining, connective tissue typically stains pink or red, making it difficult to distinguish from other structural components, necessitating specialized staining methods.

[0003] Currently, special staining combined with immunohistochemistry (IHC / IF) is commonly used to stain and label collagen fibers. Special staining often involves composite staining methods such as Masson's trichrome, which stains collagen fibers, muscle fibers, and cell nuclei. In addition to conventional trichrome staining, there are also five-color staining methods that can simultaneously visualize glycoproteins, elastic fibers, collagen fibers, muscle fibers, and cell nuclei.

[0004] The most commonly used five-color staining method is the Russell-Movat five-color staining method, invented by Movat in 1955 and modified by Russell in 1972. This method uses iodine-iron hematoxylin to stain elastic fibers and cell nuclei, alcian blue to stain glycoproteins, alcohol-soluble crocin to stain collagen fibers, and ponceau-fuchsin to stain muscle fibers and cytoplasm. It plays an irreplaceable role in cardiovascular research. This method can also be used to stain cellulose and can be used to evaluate the effect of microcrystalline cellulose, a pharmaceutical tablet excipient, on embolic pulmonary hypertension.

[0005] However, the Russell-Movat five-color staining method uses at least ten reagents, including Alcian blue staining solution, amino alcohol differentiation solution, hematoxylin stock solution, iron mordant solution, iodine diluent, hematoxylin differentiation solution, scarlet stock solution, acid fuchsin stock solution, alcohol-soluble saffron staining solution, etc. The overall staining process involves 19 steps. Although the staining results are colorful, the operation is cumbersome and it is usually difficult to ensure that five colors are displayed simultaneously, which brings many inconveniences to use. On the other hand, amino alcohol, hematoxylin stock solution, and alcohol-soluble saffron solution all contain high levels of ethanol; and amino alcohol has a pungent odor, which is unfriendly to the environment and operators during the heat treatment process. In addition, alcohol-soluble saffron is a pigment extracted from the stamens of natural saffron, which is expensive. Synthetic dyes such as safranin are usually used as substitutes, but substitutes usually have difficulty in achieving good user experience and staining effects. Summary of the Invention

[0006] The invention provides a multicolor composite dyeing kit, a dyeing method and applications thereof.

[0007] In response to the many problems of current Russell-Movat and other multi-color composite staining methods, such as complex operation, high cost, and environmental and operator-unfriendlyness, the present invention optimizes and improves the reagents used in the staining process, thereby significantly improving the staining effect of the multi-color composite staining method, making it more reliable, safer, and simpler to use for tissue staining.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides a multi-color composite staining kit, the kit comprising: a mordant solution, an Alcian blue staining solution, a hematoxylin stock solution, and a water-soluble yellow counterstain solution;

[0010] The mordant solution comprises: 0.03-0.15M of an organic acid and 0.02-0.12M of an organic acid salt; the organic acid is at least one selected from citric acid and acetic acid, and the organic acid salt is at least one selected from citrate and acetate; or the mordant solution comprises: 0.01%-0.05% of disodium ethylenediaminetetraacetate and 0.001%-0.005% of sodium hydroxide;

[0011] The hematoxylin stock solution comprises: hematoxylin 0.5-3% w / v, mannitol 5-25% w / v, glycerol 40-60% v / v, citric acid 0.08-0.5% w / v, and glacial acetic acid 0.3-0.8% v / v;

[0012] The dye contained in the water-soluble yellow counterstain solution includes at least one selected from tartrazine, carotene, and lycopene.

[0013] Existing multicolor composite staining methods typically use Bouin's fixative containing picric acid to treat tissue samples, which presents issues such as poor safety and environmental friendliness. The present invention discovers that the aforementioned mordant solution can replace Bouin's fixative, effectively improving the differentiation of fiber dyeing without the addition of picric acid, acting as a fiber coloring sensitizer. It can be used as a picric acid-free mordant to replace Bouin's fixative for mordant dyeing of connective fibers such as collagen. The high ionic strength and cross-linked epitope repair capabilities of the buffer system enhance mordant dyeing of connective tissue, thereby improving the dyeing effect.

[0014] Preferably, the mordant solution of the present invention does not contain picric acid.

[0015] Preferably, the mordant liquid of the present invention uses water as solvent.

[0016] Preferably, the mordant solution comprises: 0.05-0.1M organic acid and 0.03-0.08M organic acid salt; or, the mordant solution comprises: 0.01%-0.05% disodium ethylenediaminetetraacetic acid and 0.001%-0.005% sodium hydroxide. The remaining volume is preferably made up with water.

[0017] Preferably, the hematoxylin stock solution comprises: hematoxylin 1-2% w / v, mannitol 10-20% w / v, glycerol 45-55% v / v, citric acid 0.1-0.3% w / v, glacial acetic acid 0.4-0.6% v / v. Preferably, the remaining volume is made up with water.

[0018] In some embodiments of the present invention, the organic acid is citric acid, and the organic acid salt is acetate. Preferably, the organic acid is citric acid, and the organic acid salt is sodium acetate. The mordant solution is preferably an aqueous solution of citric acid and sodium acetate, wherein the concentration of citric acid is 0.05-0.1M and the concentration of sodium acetate is 0.03-0.08M.

[0019] In some other embodiments of the present invention, the mordant solution uses water as a solvent and includes 0.01%-0.05% of disodium ethylenediaminetetraacetate (EDTA·2Na) and 0.001%-0.005% of sodium hydroxide.

[0020] Preferably, the concentration of Alcian Blue in the Alcian Blue staining solution is preferably 0.5-1%. Preferably, the solution further comprises 0.05-0.2% glacial acetic acid. Water is preferably used as the solvent.

[0021] Preferably, the hematoxylin stock solution does not include ethanol.

[0022] Hematoxylin has poor water solubility, so the hematoxylin stock solution used in the dyeing process is typically an alcoholic hematoxylin solution (e.g., a 2% hematoxylin-5% water-95% ethanol solution). This invention has developed an alcohol-free hematoxylin stock solution. This solution contains no ethanol, making it easier to prepare, store, transport, and use. Furthermore, it dissolves and disperses hematoxylin well, exhibiting advantages such as good solubility, minimal precipitation, low volatility, and a lack of irritating odor, making it easy to store and use. Furthermore, it delivers dyeing results comparable to, or even superior to, alcoholic solutions. This stock solution can replace alcoholic hematoxylin solutions in the preparation of working solutions for spandex dyeing, or in other hematoxylin dyeing methods.

[0023] Existing staining methods, such as the Russell-Movat method, use alcohol-soluble saffron staining solution (primarily composed of natural saffron) to stain collagen fibers, which is expensive and contains high levels of ethanol. The present invention discovered that the aforementioned water-soluble yellow counterstain can replace alcohol-soluble saffron for dyeing collagen fibers, significantly reducing the cost.

[0024] Preferably, the dye contained in the water-soluble yellow counterstain is tartrazine. The present invention has found that tartrazine is closer to the color of alcohol-soluble saffron dyeing than commonly used synthetic dyes (such as Safranin O), and has a better dyeing effect. It also has better water solubility, replacing alcohol-soluble formulas, resulting in less precipitation and greater ease of use.

[0025] Preferably, the water-soluble yellow counterstain solution uses water as a solvent and comprises 0.2-3% w / v tartrazine, preferably 0.3-2% w / v tartrazine.

[0026] The water-soluble yellow counterstain solution preferably further comprises a stabilizer, preferably sodium carboxymethylcellulose (preferably added in an amount of 3%-7% w / v). The stabilizer serves to stabilize the osmotic pressure and the dyeing microenvironment.

[0027] The Alcian blue staining solution contained in the above kit can stain glycoproteins, the hematoxylin stock solution is prepared with iron mordant solution and iodine diluent to make a hematoxylin working solution, which can stain elastic fibers and cell nuclei, and the water-soluble yellow counterstain solution can stain collagen fibers.

[0028] To simultaneously stain muscle fibers and cytoplasm, the kit of the present invention further comprises an acid fuchsin staining solution; the acid fuchsin staining solution comprises: 0.1-1% w / v Ponceau red, 0.1-1% w / v acid fuchsin, and an acidic pH regulator, wherein the amount of the acidic pH regulator is such as to adjust the pH of the acid fuchsin staining solution to 2.5-3.0.

[0029] Preferably, the acid fuchsin staining solution comprises: 0.1-0.5% w / v Ponceau, 0.5-0.9% w / v Acid Fuchsin, and an acidic pH adjuster, preferably glacial acetic acid (preferably used in an amount of 1-2% v / v). Glacial acetic acid primarily regulates pH and also forms a buffer ion pair with the dye cation, ensuring pH stability during use. It also increases the ionic strength of the staining solution, promoting ionization of the target substance and enhancing the staining effect.

[0030] The kit of the present invention further comprises a differentiation solution after Alcian blue staining, wherein the differentiation solution after Alcian blue staining comprises one or more selected from Tris, ethylenediamine, and ethanolamine.

[0031] Preferably, the differentiation solution after Alcian blue staining does not contain ethanol and free ammonia.

[0032] In the present invention, the post-alcian blue differentiation solution serves as an alternative to ammonia-alcohol solutions (e.g., 5% ammonia-ethanol solution) for post-alcian blue differentiation and elastic fiber sensitization. This differentiation solution is ethanol-free and free of free ammonia, allowing it to re-expose staining sites masked by alcian blue, enhancing the effectiveness of subsequent dye staining.

[0033] The differentiation solution after Alcian blue staining can be used as needed. For example, when it is necessary to specifically observe sulfated mucins without observing carboxylic mucins, the differentiation solution after Alcian blue staining can be used for treatment. When it is necessary to observe carboxylic mucins and sulfated mucins at the same time, the treatment time of the differentiation solution after Alcian blue staining can be appropriately shortened or this step can be omitted.

[0034] Preferably, the differentiation solution after Alcian blue staining comprises Tris 2%-15% w / v; or comprises ethylenediamine 2%-15% v / v.

[0035] Preferably, the differentiation solution after Alcian blue staining comprises Tris 3%-10% w / v; or comprises ethylenediamine 3%-10% v / v.

[0036] Preferably, the differentiation solution after Alcian blue staining is a 3-10% v / v ethylenediamine aqueous solution, or a 3%-10% w / v Tris aqueous solution.

[0037] In conjunction with the above-mentioned staining reagent, the kit of the present invention further comprises one or more selected from the following (1)-(4):

[0038] (1) iron mordant solution and iodine diluent are mixed with the hematoxylin stock solution for hematoxylin staining;

[0039] (2) Hematoxylin differentiation solution, used for differentiation treatment after hematoxylin staining;

[0040] (3) Phosphotungstic acid differentiation solution, used for differentiation treatment after staining with acid fuchsin staining solution;

[0041] (4) Acidic cleaning solution, used for cleaning after treatment with phosphotungstic acid differentiation solution and water-soluble yellow counterstaining solution.

[0042] The acidic cleaning solution, iron mordant solution, iodine diluent, hematoxylin differentiation solution, and phosphotungstic acid differentiation solution described above can all be reagents commonly used in the Russell-Movat five-color staining method. Those skilled in the art can directly refer to the known Russell-Movat five-color staining method reagent formula to prepare the above reagents.

[0043] Preferably, the acidic cleaning solution is an aqueous solution of an organic weak acid. The organic weak acid is selected from one or more of acetic acid, citric acid, and tartaric acid. The organic weak acid is preferably citric acid or a combination of tartaric acid and citric acid.

[0044] The iron mordant solution and the hematoxylin differentiation solution are both acidic aqueous solutions containing 0.5%-5% w / v (preferably 1.5%-3%) ferric chloride.

[0045] The iodine diluent comprises: 0.5%-3% w / v (preferably 1%-2% w / v) of elemental iodine and 1%-7% w / v (preferably 2%-5% w / v) of potassium iodide.

[0046] The phosphotungstic acid differentiation solution comprises: 0.3%-3% w / v (preferably 0.5%-2% w / v) of phosphotungstic acid.

[0047] In some embodiments of the present invention, the iron mordant solution includes: 1.5-3% w / v ferric chloride, 0.8-1.2% v / v hydrochloric acid. The iodine diluent includes: 1-2% w / v elemental iodine, 2-5% w / v potassium iodide. The hematoxylin differentiation solution includes: 1-3% w / v ferric chloride, 0.5-1% v / v hydrochloric acid. The phosphotungstic acid differentiation solution includes: 0.5-2% phosphotungstic acid. The acidic cleaning solution includes: 0.5-4% w / v organic weak acid (preferably 0.5%-1%, preferably including 0.2-0.5% w / v citric acid and 0.3-0.5% w / v tartaric acid, or including 0.5%-1% citric acid). The above reagents all use water as the solvent, and water can be used to make up the balance.

[0048] In some embodiments of the present invention, an improved Russell-Movat five-color staining kit is provided. The kit comprises a mordant solution, an Alcian blue staining solution, a post-Alcian blue staining differentiation solution, a hematoxylin stock solution, an iron mordant solution, an iodine diluent, a hematoxylin differentiation solution, an acid fuchsin staining solution, a phosphotungstic acid differentiation solution, an acidic cleaning solution, and a water-soluble yellow counterstain. This kit exhibits higher staining efficiency and clearer staining results for tissue samples containing connective fibers and glycoproteins.

[0049] The various reagents contained in the kit of the present invention may also be supplemented with auxiliary materials such as stabilizers and preservatives (such as phenoxyethanol) to ensure the stability and storage performance of the reagents.

[0050] In a second aspect, the present invention provides any of the following applications of the multicolor composite dyeing kit described above:

[0051] (1) Connective fiber staining;

[0052] (2) Composite staining of connective fibers and glycoproteins;

[0053] (3) Staining of one or more selected from glycoproteins, elastic fibers, collagen fibers, muscle fibers, cell nuclei, and cytoplasm.

[0054] In a third aspect, the present invention provides a method for multi-color composite staining of connective fibers and glycoproteins, wherein the method comprises staining a tissue sample using the multi-color composite staining kit described above.

[0055] Preferably, the multicolor composite dyeing method comprises the following steps:

[0056] (1) Immerse the tissue sections in the mordant solution;

[0057] (2) Staining the sections treated with mordant solution with Alcian blue staining solution;

[0058] (3) Mix the hematoxylin stock solution, iron mordant solution, and iodine dilution solution to obtain the hematoxylin working solution, and use the hematoxylin working solution to stain the sections;

[0059] (4) Treat the sections with hematoxylin differentiation solution;

[0060] (5) Stain the sections with acid fuchsin staining solution;

[0061] (6) First, treat the sections with phosphotungstic acid differentiation solution;

[0062] (7) Stain the sections with a water-soluble yellow counterstain.

[0063] The above method can be used as a modified Russell-Movat staining method, which has more stable staining effect, better environmental and personnel compatibility and lower use cost.

[0064] In addition to optimizing the reagents used for dyeing, the present invention readjusts the entire dyeing process, moving the heating treatment (mordant solution treatment and preheating of the differentiation solution treatment after Alcian blue dyeing) to the very beginning, centralizing the dyeing steps, and using aqueous reagents throughout the process. This results in a moderate dyeing time and a high error tolerance for the operation steps.

[0065] In the above method, if it is necessary to specifically observe sulfated mucins without observing carboxylic acid mucins, a step of treating the differentiation solution after Alcian blue staining can be added between step (2) and step (3). Alcian blue staining will stain both carboxylic acid mucins and sulfated mucins, and at the same time, to a certain extent, mask the binding sites of subsequent dyes. Depending on the need to observe sulfated mucins and carboxylic acid mucins, the treatment time of the differentiation solution after Alcian blue staining can be adjusted as appropriate or not used.

[0066] In the above method, after the treatment with the phosphotungstic acid differentiation solution and the water-soluble yellow counterstaining solution, the method further includes the step of washing with an acidic washing solution.

[0067] In the above method, the mordant treatment lasts for 1-12 hours. Preferably, the mordant treatment is performed by preheating the mordant to 55-60°C and immersing the slices in the mordant for 1-3 hours. The mordant is a non-corrosive aqueous solution and can be preheated in a water bath, oven, or microwave oven.

[0068] In the above method, the staining time for Alcian blue staining solution is 15-25 minutes (preferably 20-25 minutes). The staining time for hematoxylin working solution is 8-30 minutes (preferably 8-15 minutes). The staining time for acid fuchsin staining solution is 1-5 minutes (preferably 3-5 minutes). The staining time for water-soluble yellow counterstain is 3-10 minutes.

[0069] In the above method, the hematoxylin stock solution, the iron mordant solution, and the iodine dilution solution are mixed in a volume ratio of 3:1-2:0.5-1.5.

[0070] In the above method, the treatment time for the hematoxylin differentiation solution is 1-3 minutes, the treatment time for the phosphotungstic acid differentiation solution is 3-8 minutes, and the treatment time for the differentiation solution after Alcian blue staining is 5-20 minutes.

[0071] In the above method, a water washing step is provided after the treatments in steps (2) to (4) and (5). An acid cleaning solution cleaning step is provided after steps (6) to (7), wherein the acid cleaning solution cleaning step is performed for 4 to 6 minutes after step (6), and the acid cleaning solution cleaning step is performed for 0.5 to 2 minutes after step (7).

[0072] In the above method, after step (7), excess water is removed, the slices are dried, and the slices are sealed after being transparentized with xylene.

[0073] In the above method, the slices are preferably frozen, paraffin, carbon wax, or plastic-sealed slices after fixation and dehydration.

[0074] Preferably, the tissue is freshly obtained and fully fixed and dehydrated before preparing paraffin or frozen sections; the sections are returned to purified water (paraffin sections are dewaxed and rehydrated, frozen sections are rewarmed and rehydrated) and then treated with mordant solution.

[0075] In a fourth aspect, the present invention provides a hematoxylin stock solution, which uses water as a solvent and includes: the hematoxylin stock solution uses water as a solvent and includes: 0.5-3% w / v hematoxylin, 5-25% w / v mannitol, 40-60% v / v glycerol, 0.08-0.5% w / v citric acid, and 0.3-0.8% v / v glacial acetic acid.

[0076] In a fifth aspect, the present invention provides use of the above-mentioned hematoxylin stock solution in staining or multi-color composite staining using hematoxylin, or in preparing a kit for hematoxylin staining or multi-color composite staining.

[0077] Preferably, the staining is for staining elastic fibers and / or cell nuclei.

[0078] Preferably, the multi-color composite staining is Masson tri-color composite staining or Russell-Movat five-color staining.

[0079] In a sixth aspect, the present invention provides use of tartrazine in collagen fiber staining or Russell-Movat five-color staining, or in preparing a kit for collagen fiber staining or Russell-Movat five-color staining.

[0080] The beneficial effects of the present invention include at least: the multi-color composite staining kit provided by the present invention can not only achieve a good staining effect, but also has a high degree of differentiation in staining different tissue structures, and can effectively distinguish mucin, cell nuclei, elastic fibers, collagen fibers and muscle fibers; and the kit does not contain hazardous chemical components such as picric acid, ethanol, ammonia, etc., which greatly reduces the relevant risks and costs during production, warehousing, transportation, use and disposal, and is more in line with the needs of sustainable development.

[0081] Taking the currently commonly used Russell-Movat staining kit as a control, the kit of the present invention can effectively simplify the operation steps, reduce the operation difficulty, improve the operation error tolerance, and significantly reduce the content of dangerous reagents such as picric acid, alcohol, and ammonia in the reagent. While significantly improving the repeatability of the staining results, it is more friendly to the environment and operators and has lower costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0083] Figure 1 This is the result of staining tissue sections using the staining kit of Example 1 of the present invention.

[0084] Figure 2 This is the result of staining tissue sections using the staining kit of Example 2 of the present invention.

[0085] Figure 3 This is the result of staining tissue sections using the staining kit of Example 3 of the present invention.

[0086] Figure 4 This is the result of staining tissue sections using the staining kit of Example 4 of the present invention.

[0087] Figure 5 This is the result of staining tissue sections using the staining kit of Example 5 of the present invention.

[0088] Figure 6 This is the result of staining tissue sections using the staining kit of Comparative Example 1 of the present invention.

[0089] Figure 7 This is the result of staining tissue sections using the staining kit of Comparative Example 2 of the present invention.

[0090] Figure 8 This is the result of staining tissue sections using the staining kit of Comparative Example 3 of the present invention.

[0091] Figure 9 This is the result of staining tissue sections using the staining kit of Comparative Example 4 of the present invention.

[0092] Figure 10 This is the result of staining tissue sections using the staining kit of Comparative Example 5 of the present invention.

[0093] Figure 11 This is the result of staining tissue sections using the staining kit of Comparative Example 6 of the present invention.

[0094] In the above figures, the two result figures of each embodiment and comparative example are observation results in different fields of view; the scale is 100µm, the magnification is 10× for the eyepiece and 20× for the objective lens. DETAILED DESCRIPTION

[0095] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0096] In the following examples, the unit of mass (w) / volume (v) is g:mL, for example, 1% (w / v) is 1 g:100 mL.

[0097] Example 1

[0098] This example provides a modified Russell-Movat five-color staining kit, which includes the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0099] (1) Mordant solution: 0.1 M citric acid monohydrate, 0.08 M sodium acetate, 1% phenoxyethanol (v / v).

[0100] (2) Alcian blue staining solution: Alcian blue 8GX 1% (w / v), glacial acetic acid 0.1% (v / v).

[0101] (3) Differentiation solution after Alcian blue staining (organic amine differentiation solution): 3% ethylenediamine (v / v).

[0102] (4) Hematoxylin stock solution: hematoxylin 2% (w / v), mannitol 15% (w / v), glycerol 50% (v / v), citric acid 0.2% (w / v), glacial acetic acid 0.5% (v / v).

[0103] (5) Iron mordant solution: ferric chloride 3% (w / v), hydrochloric acid 1% (v / v).

[0104] (6) Iodine diluent: 2% (w / v) elemental iodine, 5% (w / v) potassium iodide.

[0105] (7) Hematoxylin differentiation solution: 2% (w / v) ferric chloride, 1% (v / v) hydrochloric acid.

[0106] (8) Acid fuchsin staining solution: Ponceau 0.2% (w / v), acid fuchsin 0.8% (w / v), glacial acetic acid 2% (v / v), phenoxyethanol 1% (v / v), purified water to make up the remaining volume, pH 2.5, 25°C.

[0107] (9) Phosphotungstic acid differentiation solution: 1.5% (w / v) phosphotungstic acid.

[0108] (10) Acidic cleaning solution: tartaric acid 0.3% (w / v), citric acid 0.4% (w / v).

[0109] (11) Water-soluble yellow counterstain: tartrazine 2% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0110] This embodiment also provides a staining method based on the above-mentioned improved Russell-Movat five-color staining kit, which comprises the following steps:

[0111] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0112] (2) Preheat the mordant solution to 60°C in a water bath, immerse the slices in it and continue to keep warm for 2 hours. After treatment, remove the slices and rinse with purified water for 1 minute.

[0113] (3) After mordanting, add Alcian blue staining solution to the sections for 20 minutes and then wash with purified water for 5 minutes.

[0114] (4) Preheat the organic amine differentiation solution at 60°C for 30 minutes, immerse the slices in it and continue to keep warm for 10 minutes.

[0115] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine diluent in a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (4) for 8 minutes and wash with purified water for 3 minutes.

[0116] (6) The slices treated in step (5) were treated with hematoxylin differentiation solution for 1 min and washed with tap water for 10 min.

[0117] (7) After the treatment in step (6), the sections were stained with acid fuchsin staining solution for 3 minutes and then washed with purified water for 30 seconds.

[0118] (8) After the treatment in step (7), the slices were treated with phosphotungstic acid differentiation solution for 5 minutes and then washed with acidic cleaning solution for 5 minutes.

[0119] (9) After the treatment in step (8), the sections were treated with water-soluble yellow counterstain for 3 minutes and then washed with acidic cleaning solution for 30 seconds.

[0120] (10) Shake off excess water, dry the slices, seal the slices with xylene, neutral gum or environmentally friendly transparent sealing agent, and observe under a microscope.

[0121] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 1 ): Mucin is blue-green, cell nuclei are reddish-brown, elastic fibers are tan, collagen fibers are orange-yellow, muscle fibers are pink, and the background is red to varying degrees.

[0122] Example 2

[0123] This example provides a modified Russell-Movat five-color staining kit, which includes the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0124] (1) Mordant solution: 0.1 M citric acid monohydrate, 0.08 M sodium acetate, 0.5% phenoxyethanol (v / v).

[0125] (2) Alcian blue staining solution: Alcian blue 8GX 1% (w / v), glacial acetic acid 0.1% (v / v).

[0126] (3) Differentiation solution after Alcian blue staining (organic amine differentiation solution): 10% ethylenediamine (v / v).

[0127] (4) Hematoxylin stock solution: hematoxylin 2% (w / v), mannitol 20% (w / v), glycerol 45% (v / v), citric acid 0.1% (w / v), glacial acetic acid 0.6% (v / v).

[0128] (5) Iron mordant solution: ferric chloride 3% (w / v), hydrochloric acid 1% (v / v).

[0129] (6) Iodine diluent: 2% (w / v) elemental iodine, 5% (w / v) potassium iodide.

[0130] (7) Hematoxylin differentiation solution: 3% (w / v) ferric chloride, 1% (v / v) hydrochloric acid.

[0131] (8) Acid fuchsin staining solution: Ponceau 0.3% (w / v), acid fuchsin 0.7% (w / v), glacial acetic acid 2% (v / v), phenoxyethanol 1% (v / v), purified water to make up the remaining volume, pH 2.5, 25°C.

[0132] (9) Phosphotungstic acid differentiation solution: 2% (w / v) phosphotungstic acid.

[0133] (10) Acidic cleaning solution: 0.5% (w / v) citric acid.

[0134] (11) Water-soluble yellow counterstain: tartrazine 2% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0135] This embodiment also provides a staining method based on the above-mentioned improved Russell-Movat five-color staining kit, which comprises the following steps:

[0136] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0137] (2) Preheat the mordant solution to 60°C in a water bath, immerse the slices in it and continue to keep warm for 2 hours. After treatment, remove the slices and rinse with purified water for 1 minute.

[0138] (3) After mordanting, add Alcian blue staining solution to the sections for 20 minutes and then wash with purified water for 5 minutes.

[0139] (4) Preheat the organic amine differentiation solution at 60°C for 30 minutes, immerse the slices in it and continue to keep warm for 10 minutes.

[0140] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine diluent in a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (4) for 8 minutes and wash with purified water for 3 minutes.

[0141] (6) The slices treated in step (5) were treated with hematoxylin differentiation solution for 1 min and washed with tap water for 10 min.

[0142] (7) After the treatment in step (6), the sections were stained with acid fuchsin staining solution for 3 minutes and then washed with purified water for 30 seconds.

[0143] (8) After the treatment in step (7), the slices were treated with phosphotungstic acid differentiation solution for 5 minutes and then washed with acidic cleaning solution for 5 minutes.

[0144] (9) After the treatment in step (8), the sections were treated with water-soluble yellow counterstain for 3 minutes and then washed with acidic cleaning solution for 30 seconds.

[0145] (10) Shake off excess water, dry the slices, seal the slices with xylene, neutral gum or environmentally friendly transparent sealing agent, and observe under a microscope.

[0146] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 2 ): Mucin is blue-green, cell nuclei are reddish-brown, elastic fibers are tan, collagen fibers are orange-yellow, muscle fibers are pink, and the background is red to varying degrees.

[0147] Example 3

[0148] This example provides a modified Russell-Movat five-color staining kit, which includes the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0149] (1) Mordant solution: 0.05 M citric acid monohydrate, 0.03 M sodium acetate, 0.5% phenoxyethanol (v / v).

[0150] (2) Alcian blue staining solution: Alcian blue 8GX 0.5% (w / v), glacial acetic acid 0.1% (v / v).

[0151] (3) Differentiation solution after Alcian blue staining (organic amine differentiation solution): 3% ethylenediamine (v / v).

[0152] (4) Hematoxylin stock solution: hematoxylin 1% (w / v), mannitol 10% (w / v), glycerol 55% (v / v), citric acid 0.3% (w / v), glacial acetic acid 0.4% (v / v).

[0153] (5) Iron mordant solution: ferric chloride 1.5% (w / v), hydrochloric acid 0.8% (v / v).

[0154] (6) Iodine diluent: elemental iodine 1% (w / v), potassium iodide 2% (w / v).

[0155] (7) Hematoxylin differentiation solution: 1% (w / v) ferric chloride, 0.5% (v / v) hydrochloric acid.

[0156] (8) Acid fuchsin staining solution: Ponceau 0.1% (w / v), acid fuchsin 0.9% (w / v), glacial acetic acid 1% (v / v), sodium acetate 1% (w / v), phenoxyethanol 1% (v / v), and purified water to make up the remaining volume. pH is 3.0, 25°C.

[0157] (9) Phosphotungstic acid differentiation solution: 0.5% (w / v) phosphotungstic acid.

[0158] (10) Acidic cleaning solution: tartaric acid 0.5% (w / v), citric acid 0.2% (w / v).

[0159] (11) Water-soluble yellow counterstain: tartrazine 0.3% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0160] This embodiment also provides a staining method based on the above-mentioned improved Russell-Movat five-color staining kit, which comprises the following steps:

[0161] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0162] (2) Preheat the mordant solution to 60°C in a water bath, immerse the slices in it and continue to keep warm for 12 hours. After treatment, remove the slices and rinse with purified water for 1 minute.

[0163] (3) After mordant treatment, add Alcian blue staining solution to the sections and stain for 22 minutes, then wash with purified water for 5 minutes.

[0164] (4) Preheat the organic amine differentiation solution at 60°C for 30 minutes, immerse the slices in it and continue to keep warm for 20 minutes.

[0165] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine diluent in a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (4) for 15 minutes and wash with purified water for 3 minutes.

[0166] (6) The slices treated in step (5) were treated with hematoxylin differentiation solution for 2 min and washed with tap water for 10 min.

[0167] (7) After the treatment in step (6), the sections were stained with acid fuchsin staining solution for 5 minutes and then washed with purified water for 30 seconds.

[0168] (8) After the treatment in step (7), the slices were treated with phosphotungstic acid differentiation solution for 8 minutes and then washed with acidic cleaning solution for 5 minutes.

[0169] (9) After the treatment in step (8), the sections were treated with water-soluble yellow counterstain for 10 minutes and then washed with acidic cleaning solution for 30 seconds.

[0170] (10) Shake off excess water, dry the slices, seal the slices with xylene, neutral gum or environmentally friendly transparent sealing agent, and observe under a microscope.

[0171] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 3 ): Mucin is blue-green, cell nuclei are reddish-brown, elastic fibers are tan, collagen fibers are orange-yellow, muscle fibers are pink, and the background is red to varying degrees.

[0172] Example 4

[0173] This example provides a modified Russell-Movat five-color staining kit, which includes the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0174] (1) Mordant solution: EDTA·2Na 0.02% (w / v), sodium hydroxide 0.002% (w / v).

[0175] (2) Alcian blue staining solution: Alcian blue 8GX 1% (w / v), glacial acetic acid 0.1% (v / v).

[0176] (3) Differentiation medium after Alcian blue staining (Tris differentiation medium): Tris 3% (w / v).

[0177] (4) Hematoxylin stock solution: hematoxylin 2% (w / v), mannitol 15% (w / v), glycerol 50% (v / v), citric acid 0.2% (w / v), glacial acetic acid 0.5% (v / v).

[0178] (5) Iron mordant solution: ferric chloride 3% (w / v), hydrochloric acid 1% (v / v).

[0179] (6) Iodine diluent: 2% (w / v) elemental iodine, 5% (w / v) potassium iodide.

[0180] (7) Hematoxylin differentiation solution: 2% (w / v) ferric chloride, 1% (v / v) hydrochloric acid.

[0181] (8) Acid fuchsin staining solution: Ponceau 0.2% (w / v), acid fuchsin 0.8% (w / v), glacial acetic acid 2% (v / v), phenoxyethanol 1% (v / v), pH 2.5, 25°C.

[0182] (9) Phosphotungstic acid differentiation solution: 1.5% (w / v) phosphotungstic acid.

[0183] (10) Acidic cleaning solution: tartaric acid 0.3% (w / v), citric acid 0.4% (w / v).

[0184] (11) Water-soluble yellow counterstain: tartrazine 2% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0185] This embodiment also provides a staining method based on the above-mentioned improved Russell-Movat five-color staining kit, which comprises the following steps:

[0186] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0187] (2) Preheat the mordant solution to 60°C in a water bath, immerse the slices in it and continue to keep warm for 2 hours. After treatment, remove the slices and rinse with purified water for 1 minute.

[0188] (3) After mordanting, add Alcian blue staining solution to the sections for 20 minutes and then wash with purified water for 5 minutes.

[0189] (4) Preheat the Tris differentiation solution at 60°C for 30 minutes, immerse the slices in it and continue to keep warm for 10 minutes.

[0190] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine diluent in a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (4) for 8 minutes and wash with purified water for 3 minutes.

[0191] (6) The slices treated in step (5) were treated with hematoxylin differentiation solution for 1 min and washed with tap water for 10 min.

[0192] (7) After the treatment in step (6), the sections were stained with acid fuchsin staining solution for 3 minutes and then washed with purified water for 30 seconds.

[0193] (8) After the treatment in step (7), the slices were treated with phosphotungstic acid differentiation solution for 5 minutes and then washed with acidic cleaning solution for 5 minutes.

[0194] (9) After the treatment in step (8), the sections were treated with water-soluble yellow counterstain for 3 minutes and then washed with acidic cleaning solution for 30 seconds.

[0195] (10) Shake off excess water, dry the slices, seal the slices with xylene, neutral gum or environmentally friendly transparent sealing agent, and observe under a microscope.

[0196] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 4 ): Mucin is blue-green, cell nuclei are reddish-brown, elastic fibers are tan, collagen fibers are orange-yellow, muscle fibers are pink, and the background is red to varying degrees.

[0197] Example 5

[0198] This example provides a modified Russell-Movat five-color staining kit, which includes the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0199] (1) Mordant solution: EDTA·2Na 0.04% (w / v), sodium hydroxide 0.004% (w / v).

[0200] (2) Alcian blue staining solution: Alcian blue 8GX 1% (w / v), glacial acetic acid 0.1% (v / v).

[0201] (3) Differentiation medium after Alcian blue staining (Tris differentiation medium): Tris 10% (w / v).

[0202] (4) Hematoxylin stock solution: hematoxylin 2% (w / v), mannitol 15% (w / v), glycerol 50% (v / v), citric acid 0.2% (w / v), glacial acetic acid 0.5% (v / v).

[0203] (5) Iron mordant solution: ferric chloride 3% (w / v), hydrochloric acid 1% (v / v).

[0204] (6) Iodine diluent: 2% (w / v) elemental iodine, 5% (w / v) potassium iodide.

[0205] (7) Hematoxylin differentiation solution: 2% (w / v) ferric chloride, 1% (v / v) hydrochloric acid.

[0206] (8) Acid fuchsin staining solution: Ponceau 0.2% (w / v), acid fuchsin 0.8% (w / v), glacial acetic acid 2% (v / v), phenoxyethanol 1% (v / v), pH 2.5, 25°C.

[0207] (9) Phosphotungstic acid differentiation solution: 1.5% (w / v) phosphotungstic acid.

[0208] (10) Acidic cleaning solution: tartaric acid 0.3% (w / v), citric acid 0.4% (w / v).

[0209] (11) Water-soluble yellow counterstain: tartrazine 2% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0210] This embodiment also provides a staining method based on the above-mentioned improved Russell-Movat five-color staining kit, which comprises the following steps:

[0211] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0212] (2) Preheat the mordant solution to 60°C in a water bath, immerse the slices in it and continue to keep warm for 12 hours. After treatment, remove the slices and rinse with purified water for 1 minute.

[0213] (3) After mordanting, add Alcian blue staining solution to the sections for 20 minutes and then wash with purified water for 5 minutes.

[0214] (4) Preheat the Tris differentiation solution at 60°C for 30 minutes, immerse the slices in it and continue to keep warm for 10 minutes.

[0215] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine diluent in a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (4) for 8 minutes and wash with purified water for 3 minutes.

[0216] (6) The slices treated in step (5) were treated with hematoxylin differentiation solution for 1 min and washed with tap water for 10 min.

[0217] (7) After the treatment in step (6), the sections were stained with acid fuchsin staining solution for 3 minutes and then washed with purified water for 30 seconds.

[0218] (8) After the treatment in step (7), the slices were treated with phosphotungstic acid differentiation solution for 5 minutes and then washed with acidic cleaning solution for 5 minutes.

[0219] (9) After the treatment in step (8), the sections were treated with water-soluble yellow counterstain for 3 minutes and then washed with acidic cleaning solution for 30 seconds.

[0220] (10) Shake off excess water, dry the slices, seal the slices with xylene, neutral gum or environmentally friendly transparent sealing agent, and observe under a microscope.

[0221] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 5 ): Mucin is blue-green, cell nuclei are reddish-brown, elastic fibers are tan, collagen fibers are orange-yellow, muscle fibers are pink, and the background is red to varying degrees.

[0222] Comparative Example 1

[0223] This comparative example provides a staining kit, which is composed of reagents used in the classic Russell-Movat five-color staining method, specifically including the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0224] (1) Ammonia-alcohol reagent: 10% (v / v) ammonia water, 90% (v / v) anhydrous ethanol.

[0225] (2) Bouin's reagent: 5% (v / v) acetic acid, 25% (v / v) formaldehyde solution (37%-40%), 70% (v / v) saturated picric acid aqueous solution (about 1.33% aqueous solution).

[0226] (3) 1% Alcian blue staining solution: Alcian blue 8GX 1% (w / v).

[0227] (4) 5% sodium thiosulfate solution: sodium thiosulfate 5% (w / v).

[0228] (5) 2% hematoxylin stock solution: hematoxylin 2% (w / v), 95% ethanol solution 100% (v / v).

[0229] (6) Ferric chloride solution: ferric chloride hexahydrate 2.48% (w / v), hydrochloric acid 1% (v / v).

[0230] (7) Iodine solution: 2% (w / v) elemental iodine, 4% (w / v) potassium iodide.

[0231] (8) Ponceau stock solution: Crocein Scarlet MOO 3B (5413-75-2) 1% (w / v), acetic acid 0.5% (v / v).

[0232] (9) Acid fuchsin stock solution: acid fuchsin 0.1% (w / v), acetic acid 0.5% (v / v).

[0233] (10) 5% phosphotungstic acid solution: 5% phosphotungstic acid.

[0234] (11) 1% acetic acid solution: 1% acetic acid (v / v).

[0235] (12) Alcoholic saffron solution: saffron 6% (w / v), anhydrous ethanol 100% (v / v).

[0236] This comparative example also provides a dyeing method based on the above-mentioned dyeing kit, which is a classic Russell-Movat five-color dyeing method, comprising the following steps:

[0237] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0238] (2) Preheat the ammonia-alcohol solution to 60°C in a thermostat or water bath for 20 minutes.

[0239] (3) Immerse the rehydrated sections from step (1) in Bouin's reagent, cover, and microwave on medium for 45 seconds. Remove from the microwave and allow to soak in Bouin's reagent for 10 minutes. Do not discard the waste solution; rinse directly with cold running water for 10 minutes.

[0240] (4) Treat the sections with 5% sodium thiosulfate solution for 5 minutes and rinse with cold running water for 5 minutes.

[0241] (5) Use 1% Alcian blue staining solution to stain the slices treated in step (4) for 20 minutes. Do not discard the waste liquid, but rinse directly with cold running water for 2-5 minutes.

[0242] (6) Place the slices in the ammonia-alcohol solution and continue to heat in a water bath or incubator at 60°C for 10 minutes. Rinse with cold running water for 2-5 minutes.

[0243] (7) Mix 2% hematoxylin stock solution, ferric chloride solution, and iodine solution in a volume ratio of 3:2:1 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (6) for 60 minutes, wash them thoroughly with cold running water, and then rinse them twice with distilled water.

[0244] (8) Mix the Ponceau stock solution and the acid fuchsin stock solution in a volume ratio of 4:1 to prepare a ready-to-use Ponceau fuchsin staining working solution. Stain the sections treated in step (7) for 1 minute and rinse with distilled water twice.

[0245] (9) Add 5% phosphotungstic acid solution and treat for 5 minutes, and then 1% acetic acid solution and treat for 5 minutes. Rinse thoroughly with distilled water.

[0246] (10) The slices processed in step (9) were dehydrated by soaking in 95% ethanol once and then by soaking in anhydrous ethanol twice, each time for 1 min.

[0247] (11) Immerse the slices in alcohol saffron solution for 20 minutes. Do not discard the waste liquid. Soak them directly in anhydrous ethanol and wash them twice, each time for 1 minute.

[0248] (12) Seal the sections with xylene, neutral gum or environmentally friendly transparent sealing agent and observe under a microscope.

[0249] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 6 ): Mucin is colored green, cell nuclei are colored purple-blue, elastic fibers are colored tan, collagen fibers are colored light yellow, muscle fibers are colored pink, and the background is colored varying degrees of red. The reagent combination used in this comparative example involves specially controlled hazardous chemicals, posing safety risks during storage, experimentation, and disposal, as well as special handling costs. Furthermore, the natural raw material saffron used is relatively expensive.

[0250] Comparative Example 2

[0251] This comparative example provides a dyeing kit comprising the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0252] (1) Ammonia-alcohol reagent: 10% (v / v) ammonia water, 90% (v / v) anhydrous ethanol.

[0253] (2) Bouin's reagent: 5% (v / v) acetic acid, 25% (v / v) formaldehyde solution (37%-40%), 70% (v / v) saturated picric acid aqueous solution (about 1.33% aqueous solution).

[0254] (3) 1% Alcian blue staining solution: Alcian blue 8GX 1% (w / v).

[0255] (4) 5% sodium thiosulfate solution: sodium thiosulfate 5% (w / v).

[0256] (5) 2% hematoxylin stock solution: hematoxylin 2% (w / v), 95% ethanol solution 100% (v / v).

[0257] (6) Ferric chloride solution: ferric chloride hexahydrate 2.48% (w / v), hydrochloric acid 1% (v / v).

[0258] (7) Iodine solution: 2% (w / v) elemental iodine, 4% (w / v) potassium iodide.

[0259] (8) Ponceau stock solution: Crocein Scarlet MOO 3B (5413-75-2) 1% (w / v), acetic acid 0.5% (v / v).

[0260] (9) Acid fuchsin stock solution: acid fuchsin 0.1% (w / v), acetic acid 0.5% (v / v).

[0261] (10) 5% phosphotungstic acid solution: 5% phosphotungstic acid (w / v).

[0262] (11) 1% acetic acid solution: 1% acetic acid (v / v).

[0263] (12) Alcohol-soluble safranin staining solution: Safranin O 2% (w / v), anhydrous ethanol 100% (v / v).

[0264] This comparative example also provides a dyeing method based on the above-mentioned dyeing kit, which is based on the classic Russell-Movat five-color dyeing method and replaces the alcohol saffron solution dyeing step, comprising the following steps:

[0265] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0266] (2) Preheat the ammonia-alcohol solution to 60°C in a thermostat or water bath for 20 minutes.

[0267] (3) Immerse the rehydrated sections from step (1) in Bouin's reagent, cover, and microwave on medium for 45 seconds. Remove from the microwave and allow to soak in Bouin's reagent for 10 minutes. Do not discard the waste solution; rinse directly with cold running water for 10 minutes.

[0268] (4) Treat the sections with 5% sodium thiosulfate solution for 5 minutes and rinse with cold running water for 5 minutes.

[0269] (5) Use 1% Alcian blue staining solution to stain the slices treated in step (4) for 20 minutes. Do not discard the waste liquid, but rinse directly with cold running water for 2-5 minutes.

[0270] (6) Place the slices in the ammonia-alcohol solution and continue to heat in a water bath or incubator at 60°C for 10 minutes. Rinse with cold running water for 2-5 minutes.

[0271] (7) Mix 2% hematoxylin stock solution, ferric chloride solution, and iodine solution in a volume ratio of 3:2:1 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (6) for 60 minutes, wash them thoroughly with cold running water, and then rinse them twice with distilled water.

[0272] (8) Mix the Ponceau stock solution and the acid fuchsin stock solution in a volume ratio of 4:1 to prepare a ready-to-use Ponceau fuchsin staining working solution. Stain the sections treated in step (7) for 1 minute and rinse with distilled water twice.

[0273] (9) Add 5% phosphotungstic acid solution and treat for 5 minutes, and then 1% acetic acid solution and treat for 5 minutes. Rinse thoroughly with distilled water.

[0274] (10) The slices processed in step (9) were dehydrated by soaking in 95% ethanol once and then by soaking in anhydrous ethanol twice, each time for 1 min.

[0275] (11) Immerse the sections in alcohol-soluble safranin staining solution for 20 minutes. Do not discard the waste liquid. Immerse the sections directly in anhydrous ethanol and wash them twice, each time for 1 minute.

[0276] (12) Seal the sections with xylene, neutral gum or environmentally friendly transparent sealing agent and observe under a microscope.

[0277] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 7 ): mucin is purple-red, cell nuclei are reddish-brown, elastic fibers are tan, collagen fibers are pink, muscle fibers are red, and the background is red to varying degrees. The reagent combination used in this comparative example contains specially controlled hazardous chemicals, posing safety risks during storage, experimentation, and disposal, and requiring special handling costs. In addition, this method cannot accurately distinguish between cell nuclei, collagen fibers, and muscle fibers, indicating that using commonly used saffron analogs such as Safranin O instead of alcohol saffron solution is not effective in staining.

[0278] Comparative Example 3

[0279] This comparative example provides a staining kit, which is a commercially available staining kit and includes the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0280] (1) Mordant solution: 0.05 M EDTA, 0.08 M EDTA sodium salt, 2% (v / v) glycerol, 0.5% (w / v) sodium acetate.

[0281] (2) Alcian blue staining solution: Alcian blue 8GX 0.5% (w / v), glacial acetic acid 0.05% (v / v).

[0282] (3) Tris differentiation medium: Tris(hydroxymethyl)aminomethane 10% (w / v), PEG400 5% (v / v).

[0283] (4) Hematoxylin stock solution: hematoxylin 1% (w / v), glycerol 80% (v / v), ethylhexylglycerol 2% (v / v).

[0284] (5) Iron mordant solution: ferric chloride 1.5% (w / v), hydrochloric acid 1% (v / v).

[0285] (6) Iodine diluent: elemental iodine 1% (w / v), potassium iodide 2% (w / v).

[0286] (7) Hematoxylin differentiation solution: 0.2% (w / v) ferric chloride, 0.2% (v / v) hydrochloric acid.

[0287] (8) Acid fuchsin staining solution: Ponceau red 0.8% (w / v), acid fuchsin 0.2% (w / v), glacial acetic acid 0.5% (v / v), sodium acetate 0.5% (w / v).

[0288] (9) Phosphotungstic acid differentiation solution: 0.5% (w / v) phosphotungstic acid.

[0289] (10) Acidic cleaning solution: 0.5% (w / v) citric acid.

[0290] (11) Alcohol-soluble safranin staining solution: Safranin O 2% (w / v), anhydrous ethanol 100% (v / v).

[0291] This comparative example also provides a dyeing method based on the above-mentioned dyeing kit, which is a matching method of the commercially available commonly used dyeing kit, comprising the following steps:

[0292] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0293] (2) Immerse the rehydrated sections in the mordant solution, cover, and microwave on medium for 45 seconds. Remove from the microwave and allow to soak in the mordant solution for 10 minutes. Do not discard the waste solution; rinse directly with cold running water for 10 minutes.

[0294] (3) After mordant treatment, add Alcian blue staining solution to the slices and stain for 20 minutes. Do not discard the waste liquid, but rinse directly with cold running water for 2-5 minutes.

[0295] (4) Preheat Tris differentiation solution at 60°C for 20 minutes, place the slices in Tris differentiation solution, and continue to heat in a water bath or incubator at 60°C for 10 minutes. Rinse with cold running water for 2-5 minutes.

[0296] (5) Mix 1% hematoxylin stock solution, iron mordant solution, and iodine diluent in a volume ratio of 3:2:1 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (4) for 60 minutes, wash thoroughly with cold running water, and then rinse twice with distilled water.

[0297] (6) Treat with hematoxylin differentiation solution for 1 minute and wash with tap water for 10 minutes.

[0298] (7) Stain with acid fuchsin solution for 1 min and rinse twice with distilled water.

[0299] (8) Add phosphotungstic acid decomposition solution for 5 minutes, and then acid cleaning solution for 5 minutes. Rinse thoroughly with distilled water.

[0300] (9) The slices processed in step (8) were dehydrated by soaking in 95% ethanol once and then in anhydrous ethanol twice, each time for 1 min.

[0301] (10) Immerse the slices treated in step (9) in alcohol-soluble safranin staining solution for 20 minutes. Do not discard the waste liquid. Immerse them directly in anhydrous ethanol for 2 times, each time for 1 minute.

[0302] (11) The slices processed in step (10) are transparentized with xylene and sealed with neutral gum.

[0303] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 8 ): Mucin is blue, cell nuclei are reddish-brown, elastic fibers are tan, collagen fibers are pink, myofibers are pink, and the background is colored in varying shades of red. The reagent combination used in this comparative example involves specially controlled hazardous chemicals, posing safety risks during storage, experimentation, and disposal, and incurring special handling costs. Furthermore, this method cannot accurately distinguish cell nuclei, collagen fibers, and myofibers.

[0304] Comparative Example 4

[0305] This comparative example provides a dyeing kit, which differs from the kit of Example 3 in that the formula of the mordant solution is different, and specifically includes the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0306] (1) Mordant solution: disodium hydrogen phosphate dodecahydrate 1.64% (w / v), sodium dihydrogen phosphate dihydrate 0.45% (w / v), sodium chloride 0.85% (w / v), phenoxyethanol 0.5% (v / v).

[0307] (2) Alcian blue staining solution: Alcian blue 8GX 0.5% (w / v), glacial acetic acid 0.1% (v / v).

[0308] (3) Differentiation solution after Alcian blue staining (organic amine differentiation solution): 3% ethylenediamine (v / v).

[0309] (4) Hematoxylin stock solution: hematoxylin 1% (w / v), mannitol 10% (w / v), glycerol 55% (v / v), citric acid 0.3% (w / v), glacial acetic acid 0.4% (v / v).

[0310] (5) Iron mordant solution: ferric chloride 1.5% (w / v), hydrochloric acid 0.8% (v / v).

[0311] (6) Iodine diluent: elemental iodine 1% (w / v), potassium iodide 2% (w / v).

[0312] (7) Hematoxylin differentiation solution: 1% (w / v) ferric chloride, 0.5% (v / v) hydrochloric acid.

[0313] (8) Acid fuchsin staining solution: Ponceau red 0.1% (w / v), acid fuchsin 0.9% (w / v), glacial acetic acid 1% (v / v), sodium acetate 1% (w / v), phenoxyethanol 1% (v / v).

[0314] (9) Phosphotungstic acid differentiation solution: 0.5% (w / v) phosphotungstic acid.

[0315] (10) Acidic cleaning solution: tartaric acid 0.5% (w / v), citric acid 0.2% (w / v).

[0316] (11) Water-soluble yellow counterstain: tartrazine 0.3% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0317] This comparative example also provides a dyeing method based on the above-mentioned dyeing kit, which comprises the following steps:

[0318] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0319] (2) Preheat the mordant solution to 60°C in a water bath, immerse the slices in it and continue to keep warm for 2 hours. After treatment, remove the slices and rinse with purified water for 1 minute.

[0320] (3) After mordant treatment, add Alcian blue staining solution to the sections and stain for 22 minutes, then wash with purified water for 5 minutes.

[0321] (4) Preheat the organic amine differentiation solution to 60°C for 30 minutes, immerse the slices in it and continue to keep warm for 20 minutes.

[0322] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine diluent in a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (4) for 15 minutes and wash with purified water for 3 minutes.

[0323] (6) The slices treated in step (5) were treated with hematoxylin differentiation solution for 2 min and washed with tap water for 10 min.

[0324] (7) After the treatment in step (6), the sections were stained with acid fuchsin staining solution for 5 minutes and then washed with purified water for 30 seconds.

[0325] (8) After the treatment in step (7), the slices were treated with phosphotungstic acid differentiation solution for 8 minutes and then washed with acidic cleaning solution for 5 minutes.

[0326] (9) After the treatment in step (8), the sections were treated with water-soluble yellow counterstain for 10 minutes and then washed with acidic cleaning solution for 30 seconds.

[0327] (10) Shake off excess water, dry the slices, seal the slices with xylene, neutral gum or environmentally friendly transparent sealing agent, and observe under a microscope.

[0328] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 9 ): mucin is blue-green, cell nuclei are red, elastic fibers are light red-brown, collagen fibers are light pink, muscle fibers are red, and the background is red to varying degrees; the overall coloring of the fibers is light, and it is impossible to accurately distinguish between cell nuclei, collagen fibers, and muscle fibers; this indicates that using common buffer solutions instead of mordant solutions is not effective.

[0329] Comparative Example 5

[0330] This comparative example provides a staining kit, which differs from the kit of Example 3 in that the formula of the differentiation solution after Alcian blue staining is different, and specifically includes the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0331] (1) Mordant solution: 0.05 M citric acid monohydrate, 0.03 M sodium acetate, 0.5% phenoxyethanol (v / v).

[0332] (2) Alcian blue staining solution: Alcian blue 8GX 0.5% (w / v), glacial acetic acid 0.1% (v / v).

[0333] (3) Differentiation solution after Alcian blue staining (alkaline alcohol differentiation solution): 2% (w / v) sodium hydroxide, 20% (v / v) purified water, 80% (v / v) anhydrous ethanol.

[0334] (4) Hematoxylin stock solution: hematoxylin 1% (w / v), mannitol 10% (w / v), glycerol 55% (v / v), citric acid 0.3% (w / v), glacial acetic acid 0.4% (v / v).

[0335] (5) Iron mordant solution: ferric chloride 1.5% (w / v), hydrochloric acid 0.8% (v / v).

[0336] (6) Iodine diluent: elemental iodine 1% (w / v), potassium iodide 2% (w / v).

[0337] (7) Hematoxylin differentiation solution: 1% (w / v) ferric chloride, 0.5% (v / v) hydrochloric acid.

[0338] (8) Acid fuchsin staining solution: Ponceau red 0.1% (w / v), acid fuchsin 0.9% (w / v), glacial acetic acid 1% (v / v), sodium acetate 1% (w / v), phenoxyethanol 1% (v / v).

[0339] (9) Phosphotungstic acid differentiation solution: 0.5% (w / v) phosphotungstic acid.

[0340] (10) Acidic cleaning solution: tartaric acid 0.5% (w / v), citric acid 0.2% (w / v).

[0341] (11) Water-soluble yellow counterstain: tartrazine 0.3% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0342] This comparative example also provides a dyeing method based on the above-mentioned dyeing kit, which comprises the following steps:

[0343] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0344] (2) Preheat the mordant solution to 60°C in a water bath, immerse the slices in it and continue to keep warm for 2 hours. After treatment, remove the slices and rinse with purified water for 1 minute.

[0345] (3) After mordant treatment, add Alcian blue staining solution to the sections and stain for 22 minutes, then wash with purified water for 5 minutes.

[0346] (4) Preheat the alkaline alcohol differentiation solution to 60°C for 30 minutes, immerse the slices in it and continue to keep warm for 20 minutes.

[0347] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine diluent in a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (4) for 15 minutes and wash with purified water for 3 minutes.

[0348] (6) The slices treated in step (5) were treated with hematoxylin differentiation solution for 2 min and washed with tap water for 10 min.

[0349] (7) After the treatment in step (6), the sections were stained with acid fuchsin staining solution for 5 minutes and then washed with purified water for 30 seconds.

[0350] (8) After the treatment in step (7), the slices were treated with phosphotungstic acid differentiation solution for 8 minutes and then washed with acidic cleaning solution for 5 minutes.

[0351] (9) After the treatment in step (8), the sections were treated with water-soluble yellow counterstain for 10 minutes and then washed with acidic cleaning solution for 30 seconds.

[0352] (10) Shake off excess water, dry the slices, seal the slices with xylene, neutral gum or environmentally friendly transparent sealing agent, and observe under a microscope.

[0353] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 10 ): mucin is colored green, cell nuclei are reddish-brown, elastic fibers are light yellow, collagen fibers are light yellow, muscle fibers are pink, and the background is colored varying degrees of red. The reagent combination used in this comparative example contains specially controlled hazardous chemicals, posing safety risks during storage, experimentation, and disposal, as well as special handling costs. Furthermore, this method produces a relatively light overall tissue staining, with no specific staining of elastic fibers. This indicates that, compared with the organic amine differentiation solution of the present invention, using a common alkaline alcohol solution instead of the ammonia-alcohol solution used in the classic method is less effective.

[0354] Comparative Example 6

[0355] This comparative example provides a staining kit, which differs from the kit of Example 3 in that the formula of the hematoxylin stock solution is different. Specifically, it includes the following reagents (the remaining volume of each reagent is supplemented with purified water):

[0356] (1) Mordant solution: 0.05 M citric acid monohydrate, 0.03 M sodium acetate, 0.5% phenoxyethanol (v / v).

[0357] (2) Alcian blue staining solution: Alcian blue 8GX 0.5% (w / v), glacial acetic acid 0.1% (v / v).

[0358] (3) Differentiation solution after Alcian blue staining (organic amine differentiation solution): 3% ethylenediamine (v / v).

[0359] (4) Hematoxylin stock solution: Hematoxylin 1% (w / v).

[0360] (5) Iron mordant solution: ferric chloride 1.5% (w / v), hydrochloric acid 0.8% (v / v).

[0361] (6) Iodine diluent: elemental iodine 1% (w / v), potassium iodide 2% (w / v).

[0362] (7) Hematoxylin differentiation solution: 1% (w / v) ferric chloride, 0.5% (v / v) hydrochloric acid.

[0363] (8) Acid fuchsin staining solution: Ponceau red 0.1% (w / v), acid fuchsin 0.9% (w / v), glacial acetic acid 1% (v / v), sodium acetate 1% (w / v), phenoxyethanol 1% (v / v).

[0364] (9) Phosphotungstic acid differentiation solution: 0.5% (w / v) phosphotungstic acid.

[0365] (10) Acidic cleaning solution: tartaric acid 0.5% (w / v), citric acid 0.2% (w / v).

[0366] (11) Water-soluble yellow counterstain: tartrazine 0.3% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0367] This comparative example also provides a dyeing method based on the above-mentioned dyeing kit, which comprises the following steps:

[0368] (1) Preheat the tissue sections at 56°C for 50 minutes, dewax twice with xylene or environmentally friendly tissue clear dewaxing solution for a total of 20 minutes; wash twice with anhydrous ethanol for a total of 8 minutes; and treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 minutes each.

[0369] (2) Preheat the mordant solution to 60°C in a water bath, immerse the slices in it and continue to keep warm for 2 hours. After treatment, remove the slices and rinse with purified water for 1 minute.

[0370] (3) After mordant treatment, add Alcian blue staining solution to the sections and stain for 22 minutes, then wash with purified water for 5 minutes.

[0371] (4) Preheat the organic amine differentiation solution to 60°C for 30 minutes, immerse the slices in it and continue to keep warm for 20 minutes.

[0372] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine diluent in a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution. Stain the sections treated in step (4) for 15 minutes and wash with purified water for 3 minutes.

[0373] (6) The slices treated in step (5) were treated with hematoxylin differentiation solution for 2 min and washed with tap water for 10 min.

[0374] (7) After the treatment in step (6), the sections were stained with acid fuchsin staining solution for 5 minutes and then washed with purified water for 30 seconds.

[0375] (8) After the treatment in step (7), the slices were treated with phosphotungstic acid differentiation solution for 8 minutes and then washed with acidic cleaning solution for 5 minutes.

[0376] (9) After the treatment in step (8), the sections were treated with water-soluble yellow counterstain for 10 minutes and then washed with acidic cleaning solution for 30 seconds.

[0377] (10) Shake off excess water, dry the slices, seal the slices with xylene, neutral gum or environmentally friendly transparent sealing agent, and observe under a microscope.

[0378] The above staining method was used to stain the paraffin sections of mouse skull (which contain mucin, elastic fibers, collagen fibers with distinct structures and stable staining results). The staining results showed that ( Figure 11 ): mucin is green, cell nuclei are red, elastic fibers are light yellow, collagen fibers are light yellow, muscle fibers are pink, and the background is red to varying degrees; the overall tissue coloring is light, and the elastic fibers and cell nuclei have no specific coloring; this indicates that the use of aqueous hematoxylin solution to replace the alcoholic hematoxylin solution in the classic method is not effective.

[0379] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An improved Russell-Movat five-color staining kit, characterized in that: The kit includes: mordant solution, Alcian blue staining solution, hematoxylin stock solution, acid fuchsin staining solution and water-soluble yellow counterstaining solution; The mordant solution comprises: 0.03-0.15M citric acid and 0.02-0.12M acetate; or the mordant solution comprises: 0.01%-0.05% disodium ethylenediaminetetraacetic acid and 0.001%-0.005% sodium hydroxide; The hematoxylin stock solution uses water as a solvent and is composed of the following components: hematoxylin 0.5-3%, mannitol 5-25%, glycerol 40-60%, citric acid 0.08-0.5%, and glacial acetic acid 0.3-0.8%; The acid fuchsin staining solution comprises: 0.1-1% Ponceau red, 0.1-1% acid fuchsin, and an acidic pH regulator, wherein the amount of the acidic pH regulator is such as to adjust the pH of the acid fuchsin staining solution to 2.5-3.0; The water-soluble yellow counterstain solution uses water as a solvent and includes: tartrazine 0.2-3%; The mordant solution, Alcian blue staining solution, hematoxylin stock solution, acid fuchsin staining solution and water-soluble yellow counterstaining solution are packaged separately.

2. The improved Russell-Movat five-color staining kit according to claim 1, characterized in that The kit further comprises a differentiation solution after Alcian blue staining, wherein the differentiation solution after Alcian blue staining comprises one or more selected from Tris, ethylenediamine, and ethanolamine.

3. The improved Russell-Movat five-color staining kit according to claim 2, characterized in that The differentiation solution after Alcian blue staining includes Tris 2%-15%; or includes ethylenediamine 2%-15%.

4. The improved Russell-Movat five-color staining kit according to any one of claims 1 to 3, characterized in that The kit further comprises one or more selected from the following (1)-(4): (1) iron mordant solution and iodine diluent are mixed with the hematoxylin stock solution for hematoxylin staining; (2) Hematoxylin differentiation solution, used for differentiation treatment after hematoxylin staining; (3) Phosphotungstic acid differentiation solution, used for differentiation treatment after staining with acid fuchsin staining solution; (4) Acidic cleaning solution, used for cleaning after treatment with phosphotungstic acid differentiation solution and water-soluble yellow counterstaining solution.

5. The improved Russell-Movat five-color staining kit according to claim 4, characterized in that The acidic cleaning solution is an aqueous solution of an organic weak acid; and / or, the iron mordant solution and the hematoxylin differentiation solution are both acidic aqueous solutions containing 0.5% to 5% ferric chloride; And / or, the iodine diluent comprises: 0.5%-3% elemental iodine and 1%-7% potassium iodide; And / or, the phosphotungstic acid differentiation solution includes: 0.3%-3% phosphotungstic acid.

6. Any of the following uses of the improved Russell-Movat five-color staining kit according to any one of claims 1 to 5: (1) Connective fiber staining; (2) Composite staining of connective fibers and glycoproteins; (3) Staining of one or more selected from glycoproteins, elastic fibers, collagen fibers, muscle fibers, and cell nuclei.

7. A multicolor composite dyeing method for connective fibers and glycoproteins, characterized in that: The method comprises staining a tissue sample using the improved Russell-Movat five-color staining kit according to any one of claims 1 to 5.

8. The method according to claim 7, characterized in that The method comprises: (1) Immerse the tissue sections in the mordant solution; (2) Staining the sections treated with mordant solution with Alcian blue staining solution; (3) Mix the hematoxylin stock solution, iron mordant solution, and iodine dilution solution to obtain the hematoxylin working solution, and use the hematoxylin working solution to stain the sections; (4) Treat the sections with hematoxylin differentiation solution; (5) Stain the sections with acid fuchsin staining solution; (6) Treat the sections with phosphotungstic acid differentiation solution; (7) Stain the sections with a water-soluble yellow counterstain.

9. The method according to claim 8, characterized in that If it is necessary to specifically observe sulfated mucins without observing carboxylic mucins, add a differentiation solution treatment step after Alcian blue staining between steps (2) and (3); And / or, after the treatment with the phosphotungstic acid differentiation solution and the water-soluble yellow counterstaining solution, the method further includes a step of washing with an acidic washing solution.

10. The method according to any one of claims 7 to 9, characterized in that The mordant treatment time is 1-12 hours; and / or, the staining time with Alcian blue staining solution is 15-25 minutes; and / or, the staining time with the hematoxylin working solution is 8-30 min; and / or, the staining time with acid fuchsin staining solution is 1-5 min; And / or, the staining time of the water-soluble yellow counterstain is 3-10 minutes.

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