Multi-color composite dyeing kit, dyeing method and application of multi-color composite dyeing kit

Abstract

The invention relates to the technical field of tissue section staining, in particular to a multicolor composite staining kit, a staining method and application of the multicolor composite staining kit. The multi-color composite staining kit provided by the invention not only can realize a better staining effect and has high discrimination on staining of different tissue structures, but also does not contain dangerous chemical components such as picric acid, ethanol, ammonia and the like, so that related risks and costs are greatly reduced in the processes of production, storage, transportation, use and discarding; in addition, by adopting the kit disclosed by the invention, the operation steps can be effectively simplified, the operation difficulty is reduced, the repeatability of a dyeing result is remarkably improved, meanwhile, the kit is more friendly to the environment and operators, the cost is lower, and the kit has a better application prospect.

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 view of the many problems existing in the current multi-color composite staining methods such as Russell-Movat, including complex operation, high cost, and being unfriendly to the environment and operators, the present invention optimizes and improves the reagents used in the staining process, thereby significantly enhancing the staining effect of the multi-color composite staining method, making it more reliable, safer, and simpler when used 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, which includes: a mordant solution, an Alcian blue staining solution, a hematoxylin stock solution, and an aqueous yellow counterstain solution; Among them, the mordant solution includes: 0.03 - 0.15 M of an organic acid, and 0.02 - 0.12 M 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; alternatively, the mordant solution includes: 0.01% - 0.05% of disodium ethylenediaminetetraacetate, and 0.001% - 0.005% of sodium hydroxide; The hematoxylin stock solution includes: 0.5 - 3% w / v of hematoxylin, 5 - 25% w / v of mannitol, 40 - 60% v / v of glycerol, 0.08 - 0.5% w / v of citric acid, and 0.3 - 0.8% v / v of glacial acetic acid; The dyes contained in the aqueous yellow counterstain solution include at least one selected from lemon yellow, carotene, and lycopene.

[0010] In the existing multi-color composite staining methods, Bouin fixative containing picric acid is usually used to treat tissue samples, which has problems such as poor safety and environmental unfriendliness. The present invention discovers that the above-mentioned mordant solution can replace Bouin fixative. Without adding picric acid, it can effectively improve the discrimination of fiber staining, play the role of a fiber coloring sensitizer, and can be used as a mordant solution without picric acid to replace Bouin fixative for mordanting connective tissues such as collagen fibers. It enhances the mordanting of connective tissue staining through the high ionic strength and cross-linking epitope repair ability in the contained buffer system, thereby improving the staining effect.

[0011] Preferably, the mordant solution of the present invention does not contain picric acid.

[0012] Preferably, the mordant solution of the present invention uses water as a solvent.

[0013] Preferably, the mordant solution includes: 0.05 - 0.1 M of an organic acid, and 0.03 - 0.08 M of an organic acid salt; alternatively, the mordant solution includes: 0.01% - 0.05% of disodium ethylenediaminetetraacetate, and 0.001% - 0.005% of sodium hydroxide. The remaining volume is preferably made up with water.

[0014] Preferably, the hematoxylin stock solution comprises: 1-2% w / v of hematoxylin, 10-20% w / v of mannitol, 45-55% v / v of glycerol, 0.1-0.3% w / v of citric acid, and 0.4-0.6% v / v of glacial acetic acid. The remaining volume is preferably made up with water.

[0015] 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 0.05-0.1 M and the concentration of sodium acetate is 0.03-0.08 M.

[0016] In some other embodiments of the present invention, the mordant solution uses water as a solvent and comprises 0.01%-0.05% of disodium ethylenediaminetetraacetate (EDTA·2Na) and 0.001%-0.005% of sodium hydroxide.

[0017] Preferably, the concentration of alcian blue in the alcian blue staining solution is preferably 0.5-1%. It preferably further contains 0.05-0.2% of glacial acetic acid. Water is preferably used as a solvent.

[0018] Preferably, the hematoxylin stock solution does not contain ethanol.

[0019] Hematoxylin has poor water solubility. Therefore, the hematoxylin stock solution used in the staining process is usually a hematoxylin alcohol solution (such as a 2% hematoxylin - 5% water - 95% ethanol solution). The present invention has developed an alcohol-free hematoxylin stock solution, which does not contain ethanol, is convenient for preparation, storage, transportation and use. Moreover, hematoxylin can be better dissolved and dispersed, having advantages such as good solubility, less precipitation, low volatility, and no pungent odor, being convenient for storage and use; and can ensure a staining effect equivalent to or even better than that of the alcohol solution. This stock solution can replace the hematoxylin alcohol solution for the preparation of the elastic fiber staining working solution, or for other hematoxylin staining methods.

[0020] In the existing staining methods such as Russell-Movat, an alcohol-soluble saffron staining solution (mainly composed of natural saffron) is used to stain collagen fibers, with a high cost and a high ethanol content in the staining solution. The present invention has found that the above-mentioned water-soluble yellow counterstaining solution can replace the alcohol-soluble saffron to stain collagen fibers, greatly reducing the use cost.

[0021] Preferably, the dye contained in the water-soluble yellow counterstaining solution is tartrazine. The present invention has found that tartrazine is closer to the color of the alcohol-soluble saffron staining and has a better staining effect compared to common synthetic dyes (such as safranin O). At the same time, it has good water solubility, replacing the alcohol-soluble formula, resulting in less precipitation and being more convenient to use.

[0022] Preferably, the water-soluble yellow counterstain solution uses water as a solvent and includes: 0.2 - 3% w / v of tartrazine. Preferably, it includes 0.3 - 2% w / v of tartrazine.

[0023] The above-mentioned water-soluble yellow counterstain solution preferably further includes a stabilizer, and the stabilizer is preferably sodium carboxymethyl cellulose (the preferred addition amount is 3% - 7% w / v). The above stabilizer plays the role of stabilizing the osmotic pressure and the staining microenvironment.

[0024] The Alcian blue staining solution contained in the above kit can stain glycoproteins. The hematoxylin stock solution, iron mordant solution and iodine dilution solution are prepared into a hematoxylin staining working solution to stain elastic fibers and cell nuclei, and the water-soluble yellow counterstain solution can stain collagen fibers.

[0025] To simultaneously achieve the staining of muscle fibers and cytoplasm, the kit of the present invention further includes an acid fuchsin staining solution; the acid fuchsin staining solution includes: 0.1 - 1% w / v of ponceau, 0.1 - 1% w / v of acid fuchsin, and an acidic pH regulator, and the dosage of the acidic pH regulator is to adjust the pH of the acid fuchsin staining solution to 2.5 - 3.0.

[0026] Preferably, the acid fuchsin staining solution includes: 0.1 - 0.5% w / v of ponceau, 0.5 - 0.9% w / v of acid fuchsin, and an acidic pH regulator. The pH regulator is preferably glacial acetic acid (the preferred dosage is 1 - 2% v / v). Glacial acetic acid mainly plays the role of adjusting the pH. In addition, it can also form a buffer ion pair with the dye cation to ensure the stability of the pH during use, and can also increase the ionic strength of the staining solution, promote the ionization of the target substance, and enhance the staining effect.

[0027] The kit of the present invention further includes an Alcian blue post-staining differentiating solution, and the Alcian blue post-staining differentiating solution includes one or more selected from Tris, ethylenediamine, and ethanolamine.

[0028] Preferably, the Alcian blue post-staining differentiating solution does not contain ethanol and free ammonia.

[0029] In the present invention, the role of the Alcian blue post-staining differentiating solution is to replace the ammonia-alcohol solution (such as a 5% ammonia-ethanol solution) for the differentiation after Alcian blue staining and the sensitization of elastic fibers. This differentiating solution has the characteristics of being ethanol-free and free-ammonia-free, and can re-expose the staining sites masked by Alcian blue, enhancing the subsequent dye staining effect.

[0030] The differentiating solution after Alcian blue staining can be used as needed. For example, when specifically observing sulfomucin without observing carboxymucin, it is treated with the differentiating solution after Alcian blue staining; when observing both carboxymucin and sulfomucin simultaneously, the treatment time with the differentiating solution after Alcian blue staining can be appropriately shortened or this step can be omitted.

[0031] Preferably, the differentiating solution after Alcian blue staining comprises 2% - 15% w / v of Tris; or comprises 2% - 15% v / v of ethylenediamine.

[0032] Preferably, the differentiating solution after Alcian blue staining comprises 3% - 10% w / v of Tris; or comprises 3% - 10% v / v of ethylenediamine.

[0033] Preferably, the differentiating solution after Alcian blue staining is an aqueous solution of ethylenediamine at 3 - 10% v / v, or an aqueous solution of Tris at 3% - 10% w / v.

[0034] In combination with the above staining reagents, the kit of the present invention further comprises one or more selected from the following (1) - (4): (1) An iron mordant solution and an iodine diluting solution, which are mixed with the hematoxylin stock solution for hematoxylin staining; (2) A hematoxylin differentiating solution for differentiating treatment after hematoxylin staining; (3) A phosphotungstic acid differentiating solution for differentiating treatment after staining with an acid fuchsin staining solution; (4) An acid cleaning solution for cleaning after treatment with the phosphotungstic acid differentiating solution and an aqueous solution of water-soluble yellow counterstain.

[0035] The above-mentioned acid cleaning solution, iron mordant solution, iodine diluting solution, hematoxylin differentiating solution, and phosphotungstic acid differentiating solution 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 reagent formulations of the Russell-Movat five-color staining method to prepare the above reagents.

[0036] Preferably, the acid cleaning solution is an aqueous solution of an organic weak acid. The organic weak acid includes one or more selected from acetic acid, citric acid, and tartaric acid. The organic weak acid is preferably citric acid or a combination of tartaric acid and citric acid.

[0037] Both the iron mordant solution and the hematoxylin differentiating solution are acidic aqueous solutions of ferric chloride at 0.5% - 5% w / v (preferably 1.5% - 3%).

[0038] The iodine diluting solution comprises: 0.5% - 3% w / v (preferably 1% - 2% w / v) of iodine and 1% - 7% w / v (preferably 2% - 5% w / v) of potassium iodide.

[0039] The phosphotungstic acid differentiation solution includes: phosphotungstic acid 0.3%-3% w / v (preferably 0.5%-2% w / v).

[0040] In some embodiments of the present invention, the iron mordant solution includes: ferric chloride 1.5-3% w / v, hydrochloric acid 0.8-1.2% v / v. The iodine dilution solution includes: iodine 1-2% w / v, potassium iodide 2-5% w / v. The hematoxylin differentiation solution includes: ferric chloride 1-3% w / v, hydrochloric acid 0.5-1% v / v. The phosphotungstic acid differentiation solution includes: phosphotungstic acid 0.5-2%. The acidic cleaning solution includes: organic weak acid 0.5-4% w / v (preferably 0.5%-1%, preferably including citric acid 0.2-0.5% w / v and tartaric acid 0.3-0.5% w / v, or including citric acid 0.5%-1%). The above reagents are all dissolved in water, and the balance is made up with water.

[0041] In some embodiments of the present invention, an improved Russell-Movat five-color staining kit is provided. The kit includes a mordant solution, an alcian blue staining solution, an alcian blue post-staining differentiation solution, a hematoxylin stock solution, an iron mordant solution, an iodine dilution solution, a hematoxylin differentiation solution, an acid fuchsin staining solution, a phosphotungstic acid differentiation solution, an acidic cleaning solution, and an aqueous yellow counterstain solution. The kit has higher staining efficiency and clearer staining effect for tissue samples containing connective fibers and glycoproteins.

[0042] In the various reagents contained in the kit of the present invention, auxiliary materials such as stabilizers and preservatives (such as phenoxyethanol) can also be added to ensure the stability and storage performance of the reagents.

[0043] In the second aspect, the present invention provides any one of the following applications of the multi-color composite staining kit described above: (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, cell nuclei, and cytoplasm.

[0044] In the third aspect, the present invention provides a multi-color composite staining method for connective fibers and glycoproteins. The method is to stain a tissue sample with the multi-color composite staining kit described above.

[0045] Preferably, the multi-color composite staining method includes the following steps: (1) Immerse the tissue section in the mordant solution for treatment; (2) Stain the section treated with the mordant solution with the alcian blue staining solution; (3) Mix the hematoxylin stock solution, the iron mordant solution, and the iodine dilution solution to obtain a hematoxylin staining working solution, and stain the section with the hematoxylin staining working solution; (4) Treat the sections with hematoxylin differentiation solution; (5) Stain the sections with acid fuchsin staining solution; (6) First, treat the sections with phosphotungstic acid differentiation solution; (7) Stain the sections with water-soluble yellow counterstain solution.

[0046] The above method can be used as an improved Russell-Movat staining method, which has a more stable staining effect, better environmental and personnel compatibility, and lower usage cost.

[0047] In addition to the optimization of the reagents used for staining, the present invention has readjusted the entire staining procedure. The heating treatment (preheating operations for mordant solution treatment and differentiation solution treatment after Alcian blue staining) is advanced to the very beginning, the staining treatment steps are concentrated together, and all intermediate steps use aqueous reagents for treatment. The staining time is moderate, and the error tolerance rate of the operation steps is high.

[0048] In the above method, if specific observation of sulfomucin is required without observing carboxymucin, a differentiation solution treatment step after Alcian blue staining is added between step (2) and step (3). Alcian blue staining will stain both carboxymucin and sulfomucin simultaneously, and to a certain extent, it will obscure the binding sites of subsequent dyes. According to the observation requirements of sulfomucin and carboxymucin, the treatment time of the differentiation solution after Alcian blue staining can be adjusted as appropriate or not used.

[0049] In the above method, after the treatment with phosphotungstic acid differentiation solution and water-soluble yellow counterstain solution, there is also a step of cleaning with an acidic cleaning solution.

[0050] In the above method, the treatment time of the mordant solution is 1 - 12 hours. Preferably, the mordant solution treatment is to preheat the mordant solution to 55 - 60 °C, and the sections are immersed in it for 1 - 3 hours. The mordant solution is an aqueous solution and has no corrosiveness, and a water bath, oven, or microwave oven can be used for preheating treatment.

[0051] In the above method, the staining time of the Alcian blue staining solution is 15 - 25 min (preferably 20 - 25 min). The staining time of the hematoxylin staining working solution is 8 - 30 min (preferably 8 - 15 min). The staining time of the acid fuchsin staining solution is 1 - 5 min (preferably 3 - 5 min). The staining time of the water-soluble yellow counterstain solution is 3 - 10 min.

[0052] In the above method, the hematoxylin stock solution, iron mordant solution, and iodine diluent are mixed according to a volume ratio of 3:1 - 2:0.5 - 1.5.

[0053] In the above method, the treatment time of hematoxylin differentiation solution is 1 - 3 min. The treatment time of phosphotungstic acid differentiation solution is 3 - 8 min. The treatment time of the differentiation solution after Alcian blue staining is 5 - 20 min.

[0054] In the above method, a water washing step is set after the treatments in steps (2)-(4) and (5). An acidic cleaning solution washing step is set after steps (6)-(7), wherein the acidic cleaning solution is washed for 4 - 6 min after step (6), and the acidic cleaning solution is washed for 0.5 - 2 min after step (7).

[0055] In the above method, after step (7), the excess moisture is removed, the section is air-dried, and then sealed after being made transparent with xylene.

[0056] In the above method, the section is preferably a frozen, paraffin, carbowax, or plastic-encapsulated section after fixation and dehydration.

[0057] Preferably, the tissue is freshly obtained, fully fixed and dehydrated, and then paraffin or frozen sections are prepared; after the sections are restored to purified water (dewaxed and rehydrated for paraffin sections, rewarmed and rehydrated for frozen sections), they are treated with a mordant solution.

[0058] Fourthly, the present invention provides a hematoxylin stock solution, which uses water as a solvent and includes: 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.

[0059] Fifthly, the present invention provides the application of the above-mentioned hematoxylin stock solution in hematoxylin staining or multicolor composite staining, or in the preparation of a kit for hematoxylin staining or multicolor composite staining.

[0060] Preferably, the staining is for elastic fibers and / or cell nuclei.

[0061] Preferably, the multicolor composite staining is Masson trichrome composite staining or Russell-Movat pentachrome staining.

[0062] Sixthly, the present invention provides the application of tartrazine in collagen fiber staining or Russell-Movat pentachrome staining, or in the preparation of a kit for collagen fiber staining or Russell-Movat pentachrome staining.

[0063] The beneficial effects of the present invention at least include: The multi-color composite staining kit provided by the present invention can not only achieve good staining effects, with high discrimination for staining different tissue structures, and can effectively distinguish mucin, cell nuclei, elastic fibers, collagen fibers, and muscle fibers; moreover, the kit does not contain dangerous chemical components such as picric acid, ethanol, and ammonia, which greatly reduces the related risks and costs during production, warehousing, transportation, use, and disposal, and is more in line with the requirements of sustainable development.

[0064] Taking the currently commonly used Russell-Movat staining kit as a control, using the kit of the present invention can effectively simplify the operation steps, reduce the operation difficulty, improve the operation error tolerance rate, greatly reduce the content of dangerous reagents such as picric acid, alcohol, and ammonia in the reagents, and is more friendly to the environment and operators while significantly improving the repeatability of the staining results and having lower costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0066] Figure 1 It is the result of staining tissue sections with the staining kit of Example 1 of the present invention.

[0067] Figure 2 It is the result of staining tissue sections with the staining kit of Example 2 of the present invention.

[0068] Figure 3 It is the result of staining tissue sections with the staining kit of Example 3 of the present invention.

[0069] Figure 4 It is the result of staining tissue sections with the staining kit of Example 4 of the present invention.

[0070] Figure 5 It is the result of staining tissue sections with the staining kit of Example 5 of the present invention.

[0071] Figure 6 It is the result of staining tissue sections with the staining kit of Comparative Example 1 of the present invention.

[0072] Figure 7 It is the result of staining tissue sections with the staining kit of Comparative Example 2 of the present invention.

[0073] Figure 8 It is the result of staining tissue sections with the staining kit of Comparative Example 3 of the present invention.

[0074] Figure 9 Results of staining tissue sections with the staining kit of Comparative Example 4 of the present invention.

[0075] Figure 10 Results of staining tissue sections with the staining kit of Comparative Example 5 of the present invention.

[0076] Figure 11 Results of staining tissue sections with the staining kit of Comparative Example 6 of the present invention.

[0077] In the above figures, the two result figures of each example and comparative example are respectively the observation results of different fields of view; the scale bar is 100 µm, and the magnification is 10× for the eyepiece and 20× for the objective lens. Detailed implementation manners

[0078] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0079] In the following examples, the unit of mass (w) / volume (v) is g:mL. For example, 1% (w / v) is 1 g:100 mL.

[0080] Example 1 This example provides an improved Russell-Movat five-color staining kit, which includes the following reagents (each reagent is made up to the remaining volume with purified water): (1) Mordant solution: citric acid monohydrate 0.1 M, sodium acetate 0.08 M, phenoxyethanol 1% (v / v).

[0081] (2) Alcian blue staining solution: Alcian blue 8GX 1% (w / v), glacial acetic acid 0.1% (v / v).

[0082] (3) Differentiating solution after Alcian blue staining (organic amine differentiating solution): ethylenediamine 3% (v / v).

[0083] (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).

[0084] (5) Iron mordant solution: ferric chloride 3% (w / v), hydrochloric acid 1% (v / v).

[0085] (6) Iodine dilution solution: 2% (w / v) iodine, 5% (w / v) potassium iodide.

[0086] (7) Hematoxylin differentiation solution: 2% (w / v) ferric chloride, 1% (v / v) hydrochloric acid.

[0087] (8) Acidic fuchsin staining solution: 0.2% (w / v) ponceau, 0.8% (w / v) acidic fuchsin, 2% (v / v) glacial acetic acid, 1% (v / v) phenoxyethanol, make up the remaining volume with purified water, pH is 2.5 at 25 °C.

[0088] (9) Phosphotungstic acid differentiation solution: 1.5% (w / v) phosphotungstic acid.

[0089] (10) Acidic cleaning solution: 0.3% (w / v) tartaric acid, 0.4% (w / v) citric acid.

[0090] (11) Water-soluble yellow counterstaining solution: 2% (w / v) tartrazine, 5% (w / v) sodium carboxymethylcellulose, 1% (v / v) phenoxyethanol.

[0091] This example also provides a staining method based on the above-mentioned improved Russell-Movat five-color staining kit, which includes the following steps: (1) Preheat the tissue section at 56 °C for 50 min, dewax twice with xylene or environmental protection tissue clearing and dewaxing solution for a total of 20 min; wash twice with absolute ethanol for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in sequence.

[0092] (2) Preheat the mordant solution to 60 °C using a water bath, immerse the section in it and continue the heat preservation treatment for 2 hours. After the treatment, take it out and wash with purified water for 1 min.

[0093] (3) Drop the Alcian blue staining solution on the section after mordant treatment and stain for 20 min, wash with purified water for 5 min.

[0094] (4) Preheat the organic amine differentiation solution at 60 °C for 30 min, immerse the section in it and continue the heat preservation treatment for 10 min.

[0095] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine dilution solution according to a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution, stain the section treated in step (4) for 8 min, and wash with purified water for 3 min.

[0096] (6) Treat the section treated in step (5) with the hematoxylin differentiation solution for 1 min, and wash with tap water for 10 min.

[0097] (7) The sections processed in step (6) are dropped with acid fuchsin staining solution and stained for 3 min, and then purified with water for 30 s.

[0098] (8)The sections processed in step (7) are dropped with phosphotungstic acid differentiation solution and treated for 5 min, and then washed with acidic cleaning solution for 5 min.

[0099] (9)The sections processed in step (8) are dropped with water-soluble yellow counterstaining solution and treated for 3 min, and then washed with acidic cleaning solution for 30 s.

[0100] (10)Excess water is shaken off, the sections are air-dried, then cleared with xylene, and mounted with neutral gum or environmentally friendly transparent mounting medium, and observed under a microscope.

[0101] The above staining method is used to perform a staining test on paraffin sections of mouse skulls (which simultaneously contain mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results). The staining results show ( Figure 1 ): Mucin is stained blue-green, cell nuclei are stained red-brown, elastic fibers are stained brown, collagen fibers are stained orange-yellow, muscle fibers are stained pink, and the background is stained red to varying degrees.

[0102] Example 2 This example provides an improved Russell-Movat five-color staining kit, which includes the following reagents (each reagent is made up to the remaining volume with purified water): (1)Mordant solution: Citric acid monohydrate 0.1 M, sodium acetate 0.08 M, phenoxyethanol 0.5% (v / v).

[0103] (2)Alcian blue staining solution: Alcian blue 8GX 1% (w / v), glacial acetic acid 0.1% (v / v).

[0104] (3)Differentiation solution after Alcian blue staining (organic amine differentiation solution): Ethylenediamine 10% (v / v).

[0105] (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).

[0106] (5)Iron mordant solution: Ferric chloride 3% (w / v), hydrochloric acid 1% (v / v).

[0107] (6)Iodine dilution solution: Iodine element 2% (w / v), potassium iodide 5% (w / v).

[0108] (7)Hematoxylin differentiation solution: Ferric chloride 3% (w / v), hydrochloric acid 1% (v / v).

[0109] (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), make up the remaining volume with purified water, pH is 2.5, at 25 °C.

[0110] (9)Phosphotungstic acid differentiation solution: phosphotungstic acid 2% (w / v).

[0111] (10)Acid cleaning solution: citric acid 0.5% (w / v).

[0112] (11)Water-soluble yellow counterstaining solution: tartrazine 2% (w / v), sodium carboxymethyl cellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0113] This example also provides a staining method based on the above improved Russell-Movat five-color staining kit, which includes the following steps: (1)Preheat the tissue section at 56 °C for 50 min, dewax twice with xylene or environmentally friendly tissue clearing and dewaxing solution for a total of 20 min; wash twice with absolute ethanol for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in sequence.

[0114] (2)Preheat the mordant solution to 60 °C using a water bath, immerse the section in it and continue the heat preservation treatment for 2 hours. After the treatment, take it out and wash with purified water for 1 min.

[0115] (3)Drop the Alcian blue staining solution on the section after mordant treatment and stain for 20 min, wash with purified water for 5 min.

[0116] (4)Preheat the organic amine differentiation solution at 60 °C for 30 min, immerse the section in it and continue the heat preservation treatment for 10 min.

[0117] (5)Mix the hematoxylin stock solution, iron mordant solution, and iodine dilution solution according to a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution, stain the section treated in step (4) for 8 min, and wash with purified water for 3 min.

[0118] (6)Treat the section treated in step (5) with hematoxylin differentiation solution for 1 min and wash with tap water for 10 min.

[0119] (7)Drop the acid fuchsin staining solution on the section treated in step (6) and stain for 3 min, wash with purified water for 30 s.

[0120] (8)Drop the phosphotungstic acid differentiation solution on the section treated in step (7) and treat for 5 min, wash with the acid cleaning solution for 5 min.

[0121] (9)The sections processed in step (8) are dripped with an aqueous yellow counterstain solution and treated for 3 min, and then washed with an acidic cleaning solution for 30 s.

[0122] (10)Excess moisture is shaken off, the sections are air-dried, cleared with xylene, and mounted with neutral gum or an environmentally friendly mounting medium for transparent sections, and then observed under a microscope.

[0123] The above staining method was used to perform a staining test on paraffin sections of mouse skulls (which simultaneously contain mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results). The staining results showed ( Figure 2 ): Mucin stained blue-green, cell nuclei stained red-brown, elastic fibers stained brown, collagen fibers stained orange-yellow, muscle fibers stained pink, and the background stained red to varying degrees.

[0124] Example 3 This example provides an improved Russell-Movat five-color staining kit, which includes the following reagents (each reagent is made up to the remaining volume with purified water): (1)Mordant solution: Citric acid monohydrate 0.05 M, sodium acetate 0.03 M, phenoxyethanol 0.5% (v / v).

[0125] (2)Alcian blue staining solution: Alcian blue 8GX 0.5% (w / v), glacial acetic acid 0.1% (v / v).

[0126] (3)Differentiating solution after Alcian blue staining (organic amine differentiating solution): Ethylenediamine 3% (v / v).

[0127] (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).

[0128] (5)Iron mordant solution: Ferric chloride 1.5% (w / v), hydrochloric acid 0.8% (v / v).

[0129] (6)Iodine dilution solution: Iodine 1% (w / v), potassium iodide 2% (w / v).

[0130] (7)Hematoxylin differentiating solution: Ferric chloride 1% (w / v), hydrochloric acid 0.5% (v / v).

[0131] (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), made up to the remaining volume with purified water, pH 3.0, 25 °C.

[0132] (9)Phosphotungstic acid differentiating solution: Phosphotungstic acid 0.5% (w / v).

[0133] (10) Acid cleaning solution: 0.5% (w / v) tartaric acid, 0.2% (w / v) citric acid.

[0134] (11) Water-soluble yellow counterstain solution: 0.3% (w / v) tartrazine, 5% (w / v) sodium carboxymethylcellulose, 1% (v / v) phenoxyethanol.

[0135] This example also provides a staining method based on the above improved Russell-Movat five-color staining kit, which includes the following steps: (1) Preheat the tissue section at 56 °C for 50 min, dewax twice with xylene or environmentally friendly tissue clearing and dewaxing solution for a total of 20 min; wash twice with absolute ethanol for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in sequence.

[0136] (2) Preheat the mordant solution to 60 °C using a water bath, immerse the section in it and continue the heat preservation treatment for 12 hours. After the treatment, take it out and wash with purified water for 1 min.

[0137] (3) Drop the Alcian blue staining solution on the section after mordant treatment and stain for 22 min, then wash with purified water for 5 min.

[0138] (4) Preheat the organic amine differentiating solution to 60 °C for 30 min, immerse the section in it and continue the heat preservation treatment for 20 min.

[0139] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine dilution solution according to a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution, stain the section treated in step (4) for 15 min, and wash with purified water for 3 min.

[0140] (6) Treat the section treated in step (5) with hematoxylin differentiating solution for 2 min, and wash with tap water for 10 min.

[0141] (7) Drop the acid fuchsin staining solution on the section treated in step (6) and stain for 5 min, then wash with purified water for 30 s.

[0142] (8) Treat the section treated in step (7) with phosphotungstic acid differentiating solution for 8 min, and wash with the acid cleaning solution for 5 min.

[0143] (9) Treat the section treated in step (8) with the water-soluble yellow counterstain solution for 10 min, and wash with the acid cleaning solution for 30 s.

[0144] (10) Drain off the excess water, air-dry the section, make it transparent with xylene, and mount the section with neutral gum or environmentally friendly transparent mounting medium, then observe under a microscope.

[0145] The above staining method was used to perform a staining test on paraffin sections of mouse skulls (which simultaneously contained mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results). The staining results showed ( Figure 3 ): Mucin stained blue-green, cell nuclei stained red-brown, elastic fibers stained brown, collagen fibers stained orange-yellow, muscle fibers stained pink, and the background stained red to varying degrees.

[0146] Example 4 This example provides an improved Russell-Movat five-color staining kit, which includes the following reagents (each reagent is made up to the remaining volume with purified water): (1) Mordant solution: EDTA·2Na 0.02% (w / v), sodium hydroxide 0.002% (w / v).

[0147] (2) Alcian blue staining solution: Alcian blue 8GX 1% (w / v), glacial acetic acid 0.1% (v / v).

[0148] (3) Differentiating solution after Alcian blue staining (Tris differentiating solution): Tris 3% (w / v).

[0149] (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).

[0150] (5) Iron mordant solution: Ferric chloride 3% (w / v), hydrochloric acid 1% (v / v).

[0151] (6) Iodine dilution solution: Iodine 2% (w / v), potassium iodide 5% (w / v).

[0152] (7) Hematoxylin differentiating solution: Ferric chloride 2% (w / v), hydrochloric acid 1% (v / v).

[0153] (8) Acid fuchsin staining solution: Ponceau red 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.

[0154] (9) Phosphotungstic acid differentiating solution: Phosphotungstic acid 1.5% (w / v).

[0155] (10) Acidic cleaning solution: Tartaric acid 0.3% (w / v), citric acid 0.4% (w / v).

[0156] (11) Aqueous yellow counterstaining solution: Tartrazine 2% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0157] This embodiment also provides a staining method based on the above-improved Russell-Movat five-color staining kit, which includes the following steps: (1) Preheat the tissue section at 56 °C for 50 min, dewax twice with xylene or an environmentally friendly tissue clearing and dewaxing solution for a total of 20 min; wash twice with absolute ethanol for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in sequence.

[0158] (2) Preheat the mordant solution to 60 °C using a water bath, immerse the section in it, and continue the heat preservation treatment for 2 hours. After the treatment, take it out and wash with purified water for 1 min.

[0159] (3) Drop the Alcian blue staining solution onto the section after mordant treatment and stain for 20 min, then wash with purified water for 5 min.

[0160] (4) Preheat the Tris differentiation solution to 60 °C for 30 min, immerse the section in it, and continue the heat preservation treatment for 10 min.

[0161] (5) Mix hematoxylin stock solution, iron mordant solution, and iodine dilution solution according to a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution, stain the section treated in step (4) for 8 min, and wash with purified water for 3 min.

[0162] (6) Treat the section treated in step (5) with hematoxylin differentiation solution for 1 min and wash with tap water for 10 min.

[0163] (7) Drop the acid fuchsin staining solution onto the section treated in step (6) and stain for 3 min, then wash with purified water for 30 s.

[0164] (8) Drop the phosphotungstic acid differentiation solution onto the section treated in step (7) and treat for 5 min, then wash with an acidic cleaning solution for 5 min.

[0165] (9) Drop the water-soluble yellow counterstaining solution onto the section treated in step (8) and treat for 3 min, then wash with an acidic cleaning solution for 30 s.

[0166] (10) Drain off the excess water, air-dry the section, make it transparent with xylene, and mount the section with neutral gum or an environmentally friendly transparent mounting medium, then observe under a microscope.

[0167] Use the above staining method to perform a staining test on paraffin sections of mouse skulls (which simultaneously contain mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results). The staining results show ( Figure 4 ) that mucin stains blue-green, cell nuclei stain reddish-brown, elastic fibers stain brownish-black, collagen fibers stain orange-yellow, muscle fibers stain pink, and the background stains red to varying degrees.

[0168] Example 5 This embodiment provides an improved Russell-Movat five-color staining kit, which includes the following reagents (each reagent is made up to the remaining volume with purified water): (1) Mordant solution: EDTA·2Na 0.04% (w / v), sodium hydroxide 0.004% (w / v).

[0169] (2) Alcian blue staining solution: Alcian blue 8GX 1% (w / v), glacial acetic acid 0.1% (v / v).

[0170] (3) Differentiating solution after Alcian blue staining (Tris differentiating solution): Tris 10% (w / v).

[0171] (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).

[0172] (5) Iron mordant solution: ferric chloride 3% (w / v), hydrochloric acid 1% (v / v).

[0173] (6) Iodine dilution solution: iodine 2% (w / v), potassium iodide 5% (w / v).

[0174] (7) Hematoxylin differentiating solution: ferric chloride 2% (w / v), hydrochloric acid 1% (v / v).

[0175] (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.

[0176] (9) Phosphotungstic acid differentiating solution: phosphotungstic acid 1.5% (w / v).

[0177] (10) Acid cleaning solution: tartaric acid 0.3% (w / v), citric acid 0.4% (w / v).

[0178] (11) Aqueous yellow counterstaining solution: tartrazine 2% (w / v), sodium carboxymethyl cellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0179] This embodiment also provides a staining method based on the above improved Russell-Movat five-color staining kit, which includes the following steps: (1) Preheat the tissue section at 56 °C for 50 min, dewax twice with xylene or environmental protection tissue clearing and dewaxing solution, for a total of 20 min; wash twice with absolute ethanol, for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in turn.

[0180] (2) Preheat the mordant solution to 60 °C using a water bath, immerse the sections in it, and continue the heat preservation treatment for 12 hours. After the treatment, take out the sections and wash them with purified water for 1 min.

[0181] (3) Drop the Alcian blue staining solution onto the sections after mordant treatment and stain for 20 min, then wash with purified water for 5 min.

[0182] (4) Preheat the Tris differentiation solution at 60 °C for 30 min, immerse the sections in it, and continue the heat preservation treatment for 10 min.

[0183] (5) Mix hematoxylin stock solution, iron mordant solution, and iodine dilution solution according to a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution, stain the sections after the treatment in step (4) for 8 min, and wash with purified water for 3 min.

[0184] (6) Treat the sections after the treatment in step (5) with hematoxylin differentiation solution for 1 min, and wash with tap water for 10 min.

[0185] (7) Drop the acid fuchsin staining solution onto the sections after the treatment in step (6) and stain for 3 min, then wash with purified water for 30 s.

[0186] (8) Drop the phosphotungstic acid differentiation solution onto the sections after the treatment in step (7) and treat for 5 min, then wash with an acidic cleaning solution for 5 min.

[0187] (9) Drop the water-soluble yellow counterstaining solution onto the sections after the treatment in step (8) and treat for 3 min, then wash with an acidic cleaning solution for 30 s.

[0188] (10) Drain off the excess water, air-dry the sections, clear them with xylene, and mount them with neutral gum or an environmentally friendly mounting medium for microscopic examination.

[0189] Use the above staining method to perform a staining test on paraffin sections of mouse skulls (which simultaneously contain mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results). The staining results show ( Figure 5 ): Mucin stains blue-green, cell nuclei stain red-brown, elastic fibers stain brown, collagen fibers stain orange-yellow, muscle fibers stain pink, and the background stains red to varying degrees.

[0190] Comparative Example 1 This comparative example provides a staining kit, which consists of reagents used in the classical Russell-Movat five-color staining method, specifically including the following reagents (each reagent is made up to the remaining volume with purified water): (1) Ammonia-alcohol reagent: 10% (v / v) ammonia water and 90% (v / v) absolute ethanol.

[0191] (2) Bouin's reagent: acetic acid 5% (v / v), formaldehyde solution (37%-40%) 25% (v / v), saturated picric acid aqueous solution (about 1.33% aqueous solution) 70% (v / v).

[0192] (3) 1% Alcian blue staining solution: Alcian blue 8GX 1% (w / v).

[0193] (4) 5% sodium thiosulfate solution: sodium thiosulfate 5% (w / v).

[0194] (5) 2% hematoxylin stock solution: hematoxylin 2% (w / v), 95% ethanol solution 100% (v / v).

[0195] (6) Ferric chloride solution: ferric chloride hexahydrate 2.48% (w / v), hydrochloric acid 1% (v / v).

[0196] (7) Iodine solution: iodine 2% (w / v), potassium iodide 4% (w / v).

[0197] (8) Ponceau stock solution: Crocein Scarlet MOO 3B (5413-75-2) 1% (w / v), acetic acid 0.5% (v / v).

[0198] (9) Acid fuchsin stock solution: acid fuchsin 0.1% (w / v), acetic acid 0.5% (v / v).

[0199] (10) 5% phosphotungstic acid solution: phosphotungstic acid 5%.

[0200] (11) 1% acetic acid solution: acetic acid 1% (v / v).

[0201] (12) Alcohol safranin solution: safranin 6% (w / v), absolute ethanol 100% (v / v).

[0202] This comparative example also provides a staining method based on the above staining kit, which is the classical Russell-Movat five-color staining method, including the following steps: (1) Preheat the tissue section at 56°C for 50 min, dewax twice with xylene or environmental tissue clearing and dewaxing solution for a total of 20 min; wash twice with absolute ethanol for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in sequence.

[0203] (2) Preheat the ammoniacal alcohol solution at 60°C for 20 min using an incubator or water bath.

[0204] (3) Immerse the rehydrated sections in step (1) in Bouin's reagent, cover, heat in a microwave oven at medium power for 45 s, then take out from the microwave oven and still soak in Bouin's reagent for 10 min while standing. Do not pour out the waste liquid, and directly rinse with cold running water for 10 min.

[0205] (4) Treat the sections with 5% sodium thiosulfate solution for 5 min, and rinse with cold running water for 5 min.

[0206] (5) Stain the sections treated in step (4) with 1% Alcian blue staining solution for 20 min. Do not pour out the waste liquid, and directly rinse with cold running water for 2 - 5 min.

[0207] (6) Place the sections in ammoniacal alcohol solution, and continue to incubate at a constant temperature of 60 °C in a water bath or incubator for 10 min. Rinse with cold running water for 2 - 5 min.

[0208] (7) Mix 2% hematoxylin stock solution, ferric chloride solution, and iodine solution evenly according to 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 min, wash thoroughly with cold running water, and then rinse twice with distilled water.

[0209] (8) Mix ponceau stock solution and acid fuchsin stock solution evenly according to 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 min, and rinse twice with distilled water.

[0210] (9) Dropwise add 5% phosphotungstic acid solution and treat for 5 min, then treat with 1% acetic acid solution for 5 min. Wash thoroughly with distilled water.

[0211] (10) Immerse the sections treated in step (9) in 95% ethanol for dehydration once and in absolute ethanol for dehydration twice, 1 min each time.

[0212] (11) Immerse the sections in alcoholic safranin solution and treat for 20 min. Do not pour out the waste liquid, and directly immerse in absolute ethanol for soaking and cleaning twice, 1 min each time.

[0213] (12) Clear the sections with xylene, and mount the sections with neutral balsam or environmentally friendly mounting medium, and observe under a microscope.

[0214] Use the above staining method to perform a staining test on paraffin sections of mouse skulls (simultaneously containing mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results). The staining results show ( Figure 6): Mucin stains green, cell nuclei stain purple-blue, elastic fibers stain brownish-yellow, collagen fibers stain light yellow, muscle fibers stain pink, and the background stains red to varying degrees. The reagent combination used in this comparative example involves special controlled hazardous chemicals, which pose safety risks during storage, experimentation, and disposal, and there are special treatment costs. In addition, the natural raw material saffron used has a high cost.

[0215] Comparative Example 2 This comparative example provides a staining kit, which includes the following reagents (each reagent is made up to the remaining volume with purified water): (1) Ammonia-alcohol reagent: 10% (v / v) ammonia water, 90% (v / v) absolute ethanol.

[0216] (2) Bouin 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).

[0217] (3) 1% Alcian blue staining solution: 1% (w / v) Alcian blue 8GX.

[0218] (4) 5% Sodium thiosulfate solution: 5% (w / v) sodium thiosulfate.

[0219] (5) 2% Hematoxylin stock solution: 2% (w / v) hematoxylin, 100% (v / v) 95% ethanol solution.

[0220] (6) Ferric chloride solution: 2.48% (w / v) ferric chloride hexahydrate, 1% (v / v) hydrochloric acid.

[0221] (7) Iodine solution: 2% (w / v) iodine, 4% (w / v) potassium iodide.

[0222] (8) Ponceau red stock solution: 1% (w / v) Crocein Scarlet MOO 3B (5413 - 75 - 2), 0.5% (v / v) acetic acid.

[0223] (9) Acid fuchsin stock solution: 0.1% (w / v) acid fuchsin, 0.5% (v / v) acetic acid.

[0224] (10) 5% Phosphotungstic acid solution: 5% (w / v) phosphotungstic acid.

[0225] (11) 1% Acetic acid solution: 1% (v / v) acetic acid.

[0226] (12) Alcohol-soluble safranin staining solution: 2% (w / v) safranin O, 100% (v / v) absolute ethanol.

[0227] This comparative example also provides a staining method based on the above staining kit, which replaces the staining step of the alcoholic safranin solution on the basis of the classical Russell-Movat five-color staining method, including the following steps: (1)Preheat the tissue section at 56 °C for 50 min, dewax twice with xylene or an environmentally friendly tissue clearing and dewaxing solution for a total of 20 min; wash twice with absolute ethanol for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in sequence.

[0228] (2)Preheat the ammonia-alcohol solution at 60 °C for 20 min using an incubator or a water bath.

[0229] (3)Immerse the rehydrated section in step (1) in Bouin's reagent, cover it, heat it in a microwave oven at medium power for 45 s, then take it out of the microwave oven and still soak it in Bouin's reagent for 10 min. Do not pour out the waste liquid, and directly rinse it with cold running water for 10 min.

[0230] (4)Treat the section with 5% sodium thiosulfate solution for 5 min and rinse it with cold running water for 5 min.

[0231] (5)Stain the section treated in step (4) with 1% Alcian blue staining solution for 20 min. Do not pour out the waste liquid, and directly rinse it with cold running water for 2 - 5 min.

[0232] (6)Place the section in the ammonia-alcohol solution and continue to incubate it at 60 °C in a water bath or an incubator for 10 min. Rinse it with cold running water for 2 - 5 min.

[0233] (7)Mix 2% hematoxylin stock solution, ferric chloride solution, and iodine solution evenly according to a volume ratio of 3:2:1 to prepare a ready-to-use hematoxylin staining working solution, stain the section treated in step (6) for 60 min, wash it thoroughly with cold running water, and then rinse it twice with distilled water.

[0234] (8)Mix ponceau stock solution and acid fuchsin stock solution evenly according to a volume ratio of 4:1 to prepare a ready-to-use ponceau-fuchsin staining working solution, stain the section treated in step (7) for 1 min, and rinse it twice with distilled water.

[0235] (9)Dropwise add 5% phosphotungstic acid solution to treat for 5 min and 1% acetic acid solution to treat for 5 min. Wash it thoroughly with distilled water.

[0236] (10)Immerse the section treated in step (9) in 95% ethanol for dehydration once and in absolute ethanol for dehydration twice, 1 min each time.

[0237] (11) Immerse the sections in an alcoholic safranin staining solution for 20 min. Without pouring off the waste liquid, directly immerse them in absolute ethanol for soaking and washing twice, 1 min each time.

[0238] (12) Clear the sections with xylene and mount them with neutral balsam or an environmentally friendly mounting medium for microscopic examination.

[0239] Using the above staining method, paraffin sections of mouse skulls (simultaneously containing mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results) were subjected to staining tests. The staining results showed ( Figure 7 ): Mucin stained purple-red, cell nuclei stained reddish-brown, elastic fibers stained brownish-tan, collagen fibers stained pink, muscle fibers stained red, and the background stained red to varying degrees. The reagent combination used in this comparative example contains special controlled hazardous chemicals, posing safety risks during storage, experiments, and disposal and requiring special treatment costs; in addition, this method cannot accurately distinguish cell nuclei, collagen fibers, and muscle fibers, indicating that the staining effect of using common saffron analogs such as safranin O to replace the alcoholic saffron solution is not good.

[0240] Comparative Example 3 This comparative example provides a staining kit, which is a commonly used commercially available staining kit and includes the following reagents (each reagent is made up to the remaining volume with purified water): (1) Mordant solution: 0.05 M ethylenediaminetetraacetic acid, 0.08 M sodium ethylenediaminetetraacetate, 2% (v / v) glycerol, 0.5% (w / v) sodium acetate.

[0241] (2) Alcian blue staining solution: 0.5% (w / v) Alcian blue 8GX, 0.05% (v / v) glacial acetic acid.

[0242] (3) Tris differentiation solution: 10% (w / v) tris(hydroxymethyl)aminomethane, 5% (v / v) PEG400.

[0243] (4) Hematoxylin stock solution: 1% (w / v) hematoxylin, 80% (v / v) glycerol, 2% (v / v) 2-ethylhexylglycerol.

[0244] (5) Iron mordant solution: 1.5% (w / v) ferric chloride, 1% (v / v) hydrochloric acid.

[0245] (6) Iodine dilution solution: 1% (w / v) iodine, 2% (w / v) potassium iodide.

[0246] (7) Hematoxylin differentiation solution: 0.2% (w / v) ferric chloride, 0.2% (v / v) hydrochloric acid.

[0247] (8)Acid fuchsin staining solution: Ponceau 0.8% (w / v), Acid fuchsin 0.2% (w / v), Glacial acetic acid 0.5% (v / v), Sodium acetate 0.5% (w / v).

[0248] (9)Phosphotungstic acid differentiation solution: Phosphotungstic acid 0.5% (w / v).

[0249] (10)Acid cleaning solution: Citric acid 0.5% (w / v).

[0250] (11)Alcohol-soluble safranin staining solution: Safranin O 2% (w / v), Absolute ethanol 100% (v / v).

[0251] This comparative example also provides a staining method based on the above staining kit. This method is a supporting method for the commonly used commercially available staining kit, including the following steps: (1)Preheat the tissue section at 56 °C for 50 min, dewax twice with xylene or environmentally friendly tissue clearing and dewaxing solution, for a total of 20 min; wash twice with absolute ethanol, for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in sequence.

[0252] (2)Immerse the rehydrated section in the mordant solution, cover it, heat it in the microwave oven at medium power for 45 s, then take it out of the microwave oven and let it stand and soak in the mordant solution for 10 min. Do not pour out the waste liquid, and directly rinse it with cold running water for 10 min.

[0253] (3)Drop the Alcian blue staining solution on the section after mordant treatment and stain for 20 min. Do not pour out the waste liquid, and directly rinse it with cold running water for 2 - 5 min.

[0254] (4)Preheat the Tris differentiation solution at 60 °C for 20 min, place the section in the Tris differentiation solution, and continue to incubate it at a constant temperature of 60 °C in a water bath or incubator for 10 min. Rinse it with cold running water for 2 - 5 min.

[0255] (5)Mix the 1% hematoxylin stock solution, iron mordant solution, and iodine dilution solution evenly according to a volume ratio of 3:2:1 to prepare the ready-to-use hematoxylin staining working solution, stain the section treated in step (4) for 60 min, wash it thoroughly with cold running water, and then rinse it twice with distilled water.

[0256] (6)Treat it with the hematoxylin differentiation solution for 1 min and wash it with tap water for 10 min.

[0257] (7)Stain it with the acid fuchsin staining solution for 1 min and rinse it twice with distilled water.

[0258] (8)Drop the phosphotungstic acid differentiation solution and treat it for 5 min, then treat it with the acid cleaning solution for 5 min. Wash it thoroughly with distilled water.

[0259] (9) Immerse the sections processed in step (8) in 95% ethanol for dehydration once and in absolute ethanol for dehydration twice, 1 min each time.

[0260] (10) Immerse the sections processed in step (9) in alcoholic safranin staining solution for 20 min. Without pouring off the waste liquid, directly immerse them in absolute ethanol for soaking and washing twice, 1 min each time.

[0261] (11) Make the sections processed in step (10) transparent with xylene and mount them with neutral balsam.

[0262] Use the above staining method to perform a staining test on paraffin sections of mouse skulls (which simultaneously contain mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results). The staining results show that ( Figure 8 ) mucin stains blue, cell nuclei stain reddish-brown, elastic fibers stain brownish-tan, collagen fibers stain pink, muscle fibers stain pink, and the background stains red to varying degrees. The reagent combination used in this comparative example involves special controlled hazardous chemicals, which pose safety risks during storage, experiments, and disposal, and there are special treatment costs; in addition, this method cannot accurately distinguish cell nuclei, collagen fibers, and muscle fibers.

[0263] Comparative Example 4 This comparative example provides a staining kit, which is different from the kit in Example 3 in the formulation of the mordant solution, specifically including the following reagents (each reagent is made up to the remaining volume with purified water): (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).

[0264] (2) Alcian blue staining solution: Alcian blue 8GX 0.5% (w / v), Glacial acetic acid 0.1% (v / v).

[0265] (3) Differentiating solution after Alcian blue staining (organic amine differentiating solution): Ethylenediamine 3% (v / v).

[0266] (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).

[0267] (5) Iron mordant solution: Ferric chloride 1.5% (w / v), Hydrochloric acid 0.8% (v / v).

[0268] (6) Iodine dilution solution: Iodine 1% (w / v), Potassium iodide 2% (w / v).

[0269] (7)Hematoxylin differentiation solution: Ferric chloride 1% (w / v), hydrochloric acid 0.5% (v / v).

[0270] (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).

[0271] (9)Phosphotungstic acid differentiation solution: Phosphotungstic acid 0.5% (w / v).

[0272] (10)Acid cleaning solution: Tartaric acid 0.5% (w / v), citric acid 0.2% (w / v).

[0273] (11)Water-soluble yellow counterstaining solution: Tartrazine 0.3% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0274] This comparative example also provides a staining method based on the above staining kit, which includes the following steps: (1)Preheat the tissue section at 56 °C for 50 min, dewax twice with xylene or environmentally friendly tissue clearing and dewaxing solution for a total of 20 min; wash twice with absolute ethanol for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in sequence.

[0275] (2)Preheat the mordant solution to 60 °C using a water bath, immerse the section in it and continue the insulation treatment for 2 hours. After the treatment, take it out and wash with purified water for 1 min.

[0276] (3)Drop the Alcian blue staining solution on the section after mordant treatment and stain for 22 min, then wash with purified water for 5 min.

[0277] (4)Preheat the organic amine differentiation solution to 60 °C for 30 min, immerse the section in it and continue the insulation treatment for 20 min.

[0278] (5)Mix the hematoxylin stock solution, iron mordant solution, and iodine dilution solution according to a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution, stain the section after step (4) treatment for 15 min, and wash with purified water for 3 min.

[0279] (6)Treat the section after step (5) with the hematoxylin differentiation solution for 2 min, and wash with tap water for 10 min.

[0280] (7)Drop the acid fuchsin staining solution on the section after step (6) treatment and stain for 5 min, then wash with purified water for 30 s.

[0281] (8)Drop the phosphotungstic acid differentiation solution on the section after step (7) treatment and treat for 8 min, then wash with the acid cleaning solution for 5 min.

[0282] (9) The sections processed in step (8) are dropped with an aqueous yellow counterstain solution and processed for 10 min, and then washed with an acidic cleaning solution for 30 s.

[0283] (10) Excess water is shaken off, the sections are air-dried, cleared with xylene, and mounted with neutral gum or an environmentally friendly mounting medium, and then observed under a microscope.

[0284] The above staining method was used to perform a staining test on paraffin sections of mouse skulls (which simultaneously contained mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results). The staining results showed ( Figure 9 ) that mucin stained blue-green, cell nuclei stained red, elastic fibers stained light red-brown, collagen fibers stained light pink, muscle fibers stained red, and the background stained red to varying degrees; the overall staining of the fibers was relatively light, and it was impossible to accurately distinguish cell nuclei, collagen fibers, and muscle fibers; indicating that using a common buffer solution to replace the mordant solution had poor effects.

[0285] Comparative Example 5 This comparative example provides a staining kit, which is different from the kit in Example 3 in that the formula of the differentiation solution after Alcian blue staining is different, and specifically includes the following reagents (each reagent is made up to the remaining volume with purified water): (1) Mordant solution: 0.05 M citric acid monohydrate, 0.03 M sodium acetate, 0.5% (v / v) phenoxyethanol.

[0286] (2) Alcian blue staining solution: 0.5% (w / v) Alcian blue 8GX, 0.1% (v / v) glacial acetic acid.

[0287] (3) Differentiation solution after Alcian blue staining (alkaline alcohol differentiation solution): 2% (w / v) sodium hydroxide, 20% (v / v) purified water, 80% (v / v) absolute ethanol.

[0288] (4) Hematoxylin stock solution: 1% (w / v) hematoxylin, 10% (w / v) mannitol, 55% (v / v) glycerol, 0.3% (w / v) citric acid, 0.4% (v / v) glacial acetic acid.

[0289] (5) Iron mordant solution: 1.5% (w / v) ferric chloride, 0.8% (v / v) hydrochloric acid.

[0290] (6) Iodine dilution solution: 1% (w / v) iodine, 2% (w / v) potassium iodide.

[0291] (7) Hematoxylin differentiation solution: 1% (w / v) ferric chloride, 0.5% (v / v) hydrochloric acid.

[0292] (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).

[0293] (9) Phosphotungstic acid differentiation solution: phosphotungstic acid 0.5% (w / v).

[0294] (10) Acid cleaning solution: tartaric acid 0.5% (w / v), citric acid 0.2% (w / v).

[0295] (11) Water-soluble yellow counterstain solution: tartrazine 0.3% (w / v), sodium carboxymethyl cellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0296] This comparative example also provides a staining method based on the above staining kit, which includes the following steps: (1) Preheat the tissue section at 56 °C for 50 min, dewax twice with xylene or environmental protection tissue clearing and dewaxing solution for a total of 20 min; wash twice with absolute ethanol for a total of 8 min; treat with 95% ethanol, 85% ethanol, 75% ethanol, and purified water for 3 min each in sequence.

[0297] (2) Preheat the mordant solution to 60 °C using a water bath, immerse the section in it and continue the heat preservation treatment for 2 hours. After the treatment, take it out and wash with purified water for 1 min.

[0298] (3) Drop the Alcian blue staining solution on the section after mordant treatment and stain for 22 min, then wash with purified water for 5 min.

[0299] (4) Preheat the alkaline alcohol differentiation solution to 60 °C for 30 min, immerse the section in it and continue the heat preservation treatment for 20 min.

[0300] (5) Mix the hematoxylin stock solution, iron mordant solution, and iodine dilution solution according to a volume ratio of 3:2:1.5 to prepare a ready-to-use hematoxylin staining working solution, stain the section after the treatment in step (4) for 15 min, and wash with purified water for 3 min.

[0301] (6) Treat the section after the treatment in step (5) with the hematoxylin differentiation solution for 2 min, and wash with tap water for 10 min.

[0302] (7) Drop the acid fuchsin staining solution on the section after the treatment in step (6) and stain for 5 min, then wash with purified water for 30 s.

[0303] (8) Drop the phosphotungstic acid differentiation solution on the section after the treatment in step (7) and treat for 8 min, then wash with the acid cleaning solution for 5 min.

[0304] (9) The sections processed in step (8) are dripped with an aqueous yellow counterstain solution and treated for 10 min, and then washed with an acidic cleaning solution for 30 s.

[0305] (10) Drain off the excess water, air-dry the sections, clear them with xylene, and mount them with neutral gum or an environmentally friendly mounting medium for transparent specimens, and then observe under a microscope.

[0306] Using the above staining method, staining tests were carried out on paraffin sections of mouse skulls (which simultaneously contain mucin, elastic fibers, and collagen fibers with distinct structures and stable staining results). The staining results showed ( Figure 10 ): Mucin stained green, cell nuclei stained reddish-brown, elastic fibers stained light yellow, collagen fibers stained light yellow, muscle fibers stained pink, and the background stained red to varying degrees. There are special controlled hazardous chemicals in the reagent combination used in this comparative example, which pose safety risks during storage, experiments, and disposal, and there are also special treatment costs; in addition, the overall staining of the tissue by this method is relatively light, and there is no specific staining of elastic fibers; it shows that compared with the organic amine differentiating solution of the present invention, using a common alkaline alcohol solution to replace the ammonia-alcohol solution in the classical method has poor effects.

[0307] Comparative Example 6 This comparative example provides a staining kit, which is different from the kit in Example 3 in the formulation of the hematoxylin stock solution, and specifically includes the following reagents (each reagent is made up to the remaining volume with purified water): (1) Mordant solution: 0.05 M citric acid monohydrate, 0.03 M sodium acetate, 0.5% (v / v) phenoxyethanol.

[0308] (2) Alcian blue staining solution: 0.5% (w / v) Alcian blue 8GX, 0.1% (v / v) glacial acetic acid.

[0309] (3) Differentiating solution after Alcian blue staining (organic amine differentiating solution): 3% (v / v) ethylenediamine.

[0310] (4) Hematoxylin stock solution: 1% (w / v) hematoxylin.

[0311] (5) Iron mordant solution: 1.5% (w / v) ferric chloride, 0.8% (v / v) hydrochloric acid.

[0312] (6) Iodine dilution solution: 1% (w / v) iodine, 2% (w / v) potassium iodide.

[0313] (7) Hematoxylin differentiating solution: 1% (w / v) ferric chloride, 0.5% (v / v) hydrochloric acid.

[0314] (8) Acid fuchsin staining solution: 0.1% (w / v) ponceau, 0.9% (w / v) acid fuchsin, 1% (v / v) glacial acetic acid, 1% (w / v) sodium acetate, 1% (v / v) phenoxyethanol.

[0315] (9) Phosphotungstic acid differentiation solution: 0.5% (w / v) phosphotungstic acid.

[0316] (10) Acidic cleaning solution: tartaric acid 0.5% (w / v), citric acid 0.2% (w / v).

[0317] (11) Water-soluble yellow counterstain: tartrazine 0.3% (w / v), sodium carboxymethylcellulose 5% (w / v), phenoxyethanol 1% (v / v).

[0318] This comparative example also provides a dyeing method based on the above-mentioned dyeing kit, which comprises the following steps: (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 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.

[0329] 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. A multi-color composite staining kit, characterized in that, The kit includes: a mordant solution, an alcian blue staining solution, a hematoxylin stock solution, and an aqueous yellow counterstain solution; Among them, the mordant solution includes: 0.03 - 0.15 M of an organic acid and 0.02 - 0.12 M 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 includes: 0.01% - 0.05% of disodium ethylenediaminetetraacetate and 0.001% - 0.005% of sodium hydroxide; The hematoxylin stock solution includes: 0.5% - 3% of hematoxylin, 5% - 25% of mannitol, 40% - 60% of glycerol, 0.08% - 0.5% of citric acid, and 0.3% - 0.8% of glacial acetic acid; The dyes contained in the aqueous yellow counterstain solution include at least one selected from tartrazine, carotene, and lycopene.

2. The multi-color composite staining kit according to claim 1, wherein The organic acid is citric acid, and the organic acid salt is acetate.

3. The multi-color composite staining kit according to claim 1, wherein The aqueous yellow counterstain solution uses water as a solvent and includes: 0.2% - 3% of tartrazine.

4. The multi-color composite staining kit according to claim 1, wherein The hematoxylin stock solution does not include ethanol.

5. The multicolor composite staining kit according to any one of claims 1 to 4, characterized in that The kit further includes an acid fuchsin staining solution; The acid fuchsin staining solution includes: 0.1% - 1% of ponceau, 0.1% - 1% of acid fuchsin, and an acidic pH regulator, and the dosage of the acidic pH regulator is to adjust the pH of the acid fuchsin staining solution to 2.5 - 3.

0.

6. The multi-color composite staining kit according to any one of claims 1 to 4, characterized in that, The kit further includes an alcian blue post - differentiation solution, and the alcian blue post - differentiation solution includes one or more selected from Tris, ethylenediamine, and ethanolamine.

7. The multi-color composite staining kit according to claim 6, characterized in that The alcian blue post - differentiation solution includes 2% - 15% of Tris; or includes 2% - 15% of ethylenediamine.

8. The multi-color composite staining kit according to any one of claims 1 to 4, characterized in that, The kit further includes one or more selected from the following (1) - (4): (1) An iron mordant solution and an iodine dilution solution, which are mixed with the hematoxylin stock solution for hematoxylin staining; (2) A hematoxylin differentiation solution for the differentiation treatment after hematoxylin staining; (3) A phosphotungstic acid differentiation solution for the differentiation treatment after staining with the acid fuchsin staining solution; (4) An acidic cleaning solution for cleaning after treatment with the phosphotungstic acid differentiation solution and the aqueous yellow counterstain solution.

9. The multi-color composite staining kit according to claim 8, characterized in that, The acidic cleaning solution is an aqueous solution of an organic weak acid; and / or, both the iron mordant solution and the hematoxylin differentiation solution are acidic aqueous solutions of 0.5% - 5% ferric chloride; and / or, the iodine dilution solution includes: 0.5% - 3% of iodine and 1% - 7% of potassium iodide; and / or, the phosphotungstic acid differentiation solution includes: 0.3% - 3% of phosphotungstic acid.

10. Any one of the following applications of the multicolor composite staining kit according to any one of claims 1 - 9: (1) Connective fiber staining; (2) Connective fiber and glycoprotein composite staining; (3) Staining of one or more selected from glycoprotein, elastic fiber, collagen fiber, muscle fiber, and cell nucleus.

11. A multi-color composite staining method for connective fibers and glycoproteins, characterized in that, The method is to stain a tissue sample using the multicolor composite staining kit according to any one of claims 1 - 9.

12. The method according to claim 11, wherein The method includes: (1) Immersing a tissue section in the mordant solution for treatment; (2) Staining the section treated with the mordant solution with the alcian blue staining solution; (3) Mix the hematoxylin stock solution, iron mordant solution and iodine dilution solution to obtain the hematoxylin staining working solution, and stain the sections with the hematoxylin staining working solution; (4) Treat the sections with the hematoxylin differentiation solution; (5) Stain the sections with the acid fuchsin staining solution; (6) Treat the sections with the phosphotungstic acid differentiation solution; (7) Stain the sections with the water-soluble yellow counterstain solution.

13. The method according to claim 12, wherein If specific observation of sulfomucin is required without observing carboxymucin, add a step of treating with the Alcian blue staining and differentiation solution between step (2) and step (3); And / or, after treatment with the phosphotungstic acid differentiation solution and the water-soluble yellow counterstain solution, it further includes a step of washing with an acidic cleaning solution.

14. The method according to claim 12 or 13, characterized in that The time for mordant solution treatment is 1 - 12 hours; And / or, the staining time with the Alcian blue staining solution is 15 - 25 min; And / or, the staining time with the hematoxylin staining working solution is 8 - 30 min; And / or, the staining time with the acid fuchsin staining solution is 1 - 5 min; And / or, the staining time with the water-soluble yellow counterstain solution is 3 - 10 min.

15. A hematoxylin stock solution, characterized in that, The hematoxylin stock solution uses water as a solvent and includes: 0.5 - 3% hematoxylin, 5 - 25% mannitol, 40 - 60% glycerol, 0.08 - 0.5% citric acid, 0.3 - 0.8% glacial acetic acid.

16. Application of tartrazine in collagen fiber staining or Russell - Movat pentachrome staining, or in the preparation of a kit for collagen fiber staining or Russell - Movat pentachrome staining.

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