Economical collagenous fiber dyeing kit and preparation method thereof
By modifying the dye solutions of iron hematoxylin, eosin, and fuchsin, the stability and uniformity problems of existing dyeing reagents were solved, achieving high-stability and high-contrast dyeing of collagen fibers, which is suitable for a variety of dyeing methods.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO HENGXING UNIV OF SCI & TECH
- Filing Date
- 2026-03-12
- Publication Date
- 2026-05-12
AI Technical Summary
Existing collagen fiber staining reagents suffer from problems such as oxidative browning, poor stability, uneven staining, and low contrast, which affect the clarity and reproducibility of HE staining.
The dye stability and solubility are improved by using modified iron hematoxylin, modified eosin dyeing solution and modified fuchsin solution, and the pH buffer system is optimized to ensure dyeing uniformity and contrast.
The prepared staining reagent has good stability, produces uniform staining results, high color contrast, and high image clarity, and is suitable for a variety of staining methods.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to an economical collagen fiber staining kit and its preparation method. Background Technology
[0002] Collagen fiber staining reagents are a class of chemical dyes used in pathology to specifically display collagen in tissue sections. Their core function is to give collagen fibers a bright color, making them clearly visible under a microscope. These reagents can effectively distinguish collagen fibers from other components such as muscle fibers and are mainly used to assess the degree of tissue fibrosis, assist in the differential diagnosis of tumors, and study the tissue repair process.
[0003] In existing technologies, conventional iron hematoxylin a solution is prone to oxidative browning and has poor stability, while b solution is too acidic and can easily lead to excessive tissue corrosion and uneven mordanting. Furthermore, when the two are mixed, precipitation or inconsistent staining often occurs. Ordinary eosin staining solution has unstable pH and is prone to precipitation, resulting in uneven cytoplasmic staining and residual background, which affects the clarity and reproducibility of HE staining. Commonly used fuchsin staining solution has poor compatibility with picric acid dilution solution, which can easily lead to insufficient dissolution and uneven staining, resulting in dull contrast and large fluctuations in collagen fiber staining results.
[0004] Based on this, the present invention provides an economical collagen fiber staining kit and its preparation method. Summary of the Invention
[0005] The purpose of this invention is to provide an economical collagen fiber staining kit and its preparation method. The staining reagent prepared by this invention has a long shelf life, indicating its good stability; the coefficient of variation (CV%) is low, indicating uniform staining results; and the color contrast index (ΔOD) is high, indicating that the target and background are clearly distinguished after staining, resulting in high image clarity.
[0006] To achieve the above objectives, the present invention provides the following technical solution: an economical collagen fiber staining kit and its preparation method, comprising the following reagents: modified iron hematoxylin, Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin solution, differentiation concentrate, acidic differentiation solution, bluing solution and Scott's blueing solution; The modified iron hematoxylin is prepared by mixing modified iron hematoxylin a solution and modified iron hematoxylin b solution in equal volumes at a volume ratio of 1:1. The modified fuchsin solution is prepared by mixing modified fuchsin dye solution a and modified fuchsin diluent b at a volume ratio of 1:9.
[0007] Preferably, the modified iron hematoxylin a solution is prepared as follows: hematoxylin colorant is added to anhydrous ethanol in a water bath at 25-45℃ and stirred continuously until completely dissolved; then glycerol, ascorbic acid and polyethylene glycol 400 are added sequentially, and stirring is continued for 5-10 minutes after each addition until the mixture is homogeneous; after all components have been added, the mixed solution is transferred to a light-proof container, stirred at room temperature, then sealed and allowed to stand and mature at 4℃ in the dark for 4-6 weeks to obtain the modified iron hematoxylin a solution.
[0008] Preferably, the mass ratio of hematoxylin colorant to anhydrous ethanol is 1:(75-85); the mass ratio of hematoxylin colorant to glycerol is 1:(0.6-1); the mass ratio of hematoxylin colorant to ascorbic acid is 1:(0.04-0.06); and the mass ratio of hematoxylin colorant to polyethylene glycol 400 is 1:(2-3).
[0009] Preferably, the modified iron hematoxylin b solution is prepared by adding deionized water to a 30% ferric chloride solution and stirring until homogeneous. Then, concentrated hydrochloric acid is added dropwise while continuously stirring, and the pH value is controlled at 1.5-2.5. After the addition is complete, sodium citrate and potassium aluminum sulfate powder are added in sequence, and stirring is continued until the solid is completely dissolved and the solution is clear, thus obtaining the modified iron hematoxylin b solution.
[0010] Preferably, the mass ratio of ferric chloride solution to deionized water is 1:(0.6-0.7); the mass ratio of ferric chloride solution to concentrated hydrochloric acid is 1:(0.2-0.25); the mass ratio of ferric chloride solution to sodium citrate is 1:(0.05-0.07); and the mass ratio of ferric chloride solution to potassium aluminum sulfate powder is 1:(0.02-0.04).
[0011] Preferably, the modified eosin staining solution is prepared as follows: 95-100 parts of an acetate-sodium acetate buffer solution with a pH of 4.8 are mixed with 1 part of water-soluble eosin Y powder, and the mixture is continuously stirred to form a uniform red clear liquid. 0.08-0.12 parts of polysorbate-20 and 0.4-0.6 parts of benzyl alcohol are added sequentially to the clear liquid, and the mixture is continuously stirred until the added components are fully mixed. Finally, the volume is adjusted and homogenized to obtain the modified eosin staining solution.
[0012] Preferably, the modified fuchsin dye solution a is prepared by mixing 95-105 parts of 1% acetic acid aqueous solution with 1 part of acid fuchsin powder and stirring continuously until the powder is dissolved and the solution is uniformly deep red; then, 0.02-0.03 parts of sodium benzoate and 0.08-0.12 parts of polyethylene glycol 6000 are added sequentially and stirred continuously until the added components are evenly dispersed and dissolved to obtain the modified fuchsin dye solution a.
[0013] Preferably, the modified fuchsin diluent b is prepared as follows: 1 part of saturated picric acid aqueous solution is mixed with 0.10-0.15 parts of 70% ethanol solution and stirred until fully mixed; then, under stirring conditions, 0.010-0.015 parts of glacial acetic acid solution is added to control the pH to be stable at 2.0-2.5, and then 0.007-0.009 parts of saturated sodium chloride aqueous solution is added, and the stirring speed is increased until all components are completely dissolved and the solution is clear; after mixing, the stirring speed is reduced and stirring is continued to complete homogenization, thus obtaining modified fuchsin diluent b.
[0014] Preferably, the method for preparing the saturated picric acid aqueous solution is as follows: First, add 0.06-0.08 parts of picric acid crystals to 1 part of distilled water and stir continuously until completely dissolved; then, based on 1 part of distilled water, add 0.03-0.05 parts of picric acid crystals and stir until the picric acid crystals are completely dissolved and the solution is clear, repeating this process; when solid residue is observed at the bottom of the container and stirring no longer dissolves, it indicates that saturation has been reached; stop stirring and let stand, then filter and take the supernatant to obtain the saturated picric acid aqueous solution.
[0015] An economical collagen fiber staining kit, comprising nine dropper bottles containing different reagents: modified iron hematoxylin (prepared by mixing modified iron hematoxylin a solution and modified iron hematoxylin b solution in a 1:1 volume ratio), Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin solution (prepared by mixing modified fuchsin a solution and modified fuchsin dilution b solution in a 1:9 volume ratio), differentiation concentrate, acidic differentiation solution, bluing solution, and Scott's blueing solution.
[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention significantly improves the staining performance and stability of iron hematoxylin through systematic modification. Specifically, the addition of glycerol, ascorbic acid, and polyethylene glycol 400 to the modified iron hematoxylin a solution enhances dye solubility, delays oxidative browning, and prevents precipitation, resulting in clearer and more uniform nuclear staining. The introduction of sodium citrate and potassium aluminum sulfate into the modified iron hematoxylin b solution constitutes a buffer mordant system, stabilizing iron ion release, reducing excessive corrosiveness of hydrochloric acid, and enhancing staining contrast. When used, the two solutions are mixed in a 1:1 volume ratio to ensure excellent selectivity, vivid contrast, and good reproducibility in nuclear staining, while the mixture exhibits high stability and is less prone to adverse precipitation or mutual incompatibility.
[0017] 2. This invention solves the problems of easy precipitation and uneven staining of traditional eosin staining solutions by using an acetate-sodium acetate buffer system with a pH of 4.8 instead of pure water as a solvent, and by adding polysorbate-20 and benzyl alcohol. This formula can maintain the pH stability of the staining solution, enhance the permeability and affinity to the cytoplasm, make the cytoplasm staining bright and natural, and the background clean, thus improving the quality and observation effect of hematoxylin and eosin staining series.
[0018] 3. In this invention, modified fuchsin dye solution a is prepared by adding sodium benzoate and polyethylene glycol 6000 to the acetic acid system, which improves the solubility and solution stability of acid fuchsin, resulting in more uniform dyeing and less aggregation. Modified fuchsin diluent b optimizes the compatibility and pH buffering capacity of the saturated picric acid solution by introducing ethanol, acetic acid, and sodium chloride, making it easier to mix with other components. When the two are mixed at a volume ratio of 1:9, a uniform and stable working solution can be quickly formed, achieving a dyeing effect with strong specificity and clear contrast on collagen fibers in dyeing methods such as VG. Moreover, there is no precipitation or stratification during the mixing process, ensuring the consistency and reliability of the dyeing results. Detailed Implementation
[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0020] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application are described clearly and completely.
[0021] Example 1: A method for preparing an economical collagen fiber staining reagent, comprising the following reagents: modified iron hematoxylin, Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin solution, differentiation concentrate, acidic differentiation solution, bluing solution, and Scott's blueing solution. Modified iron hematoxylin was prepared by mixing modified iron hematoxylin a solution and modified iron hematoxylin b solution in equal volumes at a ratio of 1:1. The modified fuchsin solution is prepared by mixing modified fuchsin dye solution a and modified fuchsin diluent b at a volume ratio of 1:9.
[0022] The modified iron hematoxylin a solution is prepared as follows: Hematoxylin colorant is added to anhydrous ethanol in a 25°C water bath and stirred continuously at 200 rpm until completely dissolved; then glycerol, ascorbic acid and polyethylene glycol 400 are added sequentially, and after each addition, the mixture is stirred continuously at 200 rpm for 5 minutes until homogeneous; after all components have been added, the mixed solution is transferred to a light-proof container and stirred at 200 rpm for 10 minutes at room temperature, then sealed and allowed to stand and mature at 4°C for 4 weeks in the dark to obtain the modified iron hematoxylin a solution.
[0023] The mass ratio of hematoxylin colorant to anhydrous ethanol is 1:75; the mass ratio of hematoxylin colorant to glycerol is 1:0.6; the mass ratio of hematoxylin colorant to ascorbic acid is 1:0.04; and the mass ratio of hematoxylin colorant to polyethylene glycol 400 is 1:2.
[0024] The modified iron hematoxylin b solution was prepared as follows: Under ventilated conditions and at 20°C, deionized water was added to a 30% ferric chloride solution and stirred at 150 rpm for 2 min. Then, while continuously stirring, concentrated hydrochloric acid was added dropwise at 1 mL / min, and the pH was controlled at 1.5. After the addition was complete, sodium citrate and potassium aluminum sulfate powder were added sequentially, and the stirring speed was increased to 200 rpm. The stirring was continued for 10 min until the solid was completely dissolved and the solution was clear. Finally, deionized water was added to bring the volume to the required total volume, and the solution was stirred at 100 rpm for 5 min to mix. The solution was then transferred to a brown bottle and stored at room temperature away from light to obtain the modified iron hematoxylin b solution.
[0025] The mass ratio of ferric chloride solution to deionized water is 1:0.6; the mass ratio of ferric chloride solution to concentrated hydrochloric acid is 1:0.2; the mass ratio of ferric chloride solution to sodium citrate is 1:0.05; and the mass ratio of ferric chloride solution to potassium aluminum sulfate powder is 1:0.02.
[0026] The modified eosin staining solution was prepared as follows: 95 parts of a pH 4.8 acetate-sodium acetate buffer solution and 1 part of water-soluble eosin Y powder were mixed at 20℃ and stirred at 200 rpm for 30 min until the powder was completely dissolved, forming a uniform red clear solution. 0.08 parts of polysorbate-20 and 0.4 parts of benzyl alcohol were added to the clear solution sequentially, and the stirring speed was reduced to 150 rpm. The mixture was stirred for 10 min to ensure that all added components were thoroughly mixed. Finally, the obtained staining solution was adjusted to the target volume with a pH 4.8 acetate-sodium acetate buffer solution and homogenized by stirring at 100 rpm for 5 min. The solution was then transferred to a light-protected reagent bottle and stored at room temperature to obtain the modified eosin staining solution.
[0027] The modified fuchsin dye solution a is prepared as follows: Under light-protected conditions at 20°C, 95 parts by mass of a 1% acetic acid aqueous solution are mixed with 1 part of acid fuchsin powder and stirred continuously at 200 rpm for 50 min until the powder dissolves and the solution is uniformly deep red; then, 0.02 parts by mass of sodium benzoate and 0.08 parts by mass of polyethylene glycol 6000 are added sequentially and stirred continuously at 15 min to ensure that the added components are fully dispersed and dissolved; finally, the bottle wall is washed with a 1% acetic acid solution and the volume is adjusted to the target volume, and then stirred at 150 rpm for 5 min to make the system completely homogeneous. The solution is then transferred to a light-protected container, sealed, and stored at room temperature to obtain the modified fuchsin dye solution a.
[0028] The modified fuchsin diluent b is prepared as follows: Under 20°C and light-proof and ventilated conditions, 1 part of saturated picric acid aqueous solution and 0.10 parts of 70% ethanol solution are mixed and stirred at 150 rpm for 5 min. While maintaining the stirring speed, 0.010 parts of glacial acetic acid solution are added to control the pH to be stable at 2.0, and then 0.007 parts of saturated sodium chloride aqueous solution are added. The stirring speed is increased to 200 rpm and stirred for another 15 min until all components are completely dissolved and the solution is clear. After mixing, the stirring speed is reduced to 100 rpm and stirred for 5 min to complete homogenization. The solution is then transferred to a light-proof reagent bottle, sealed, and stored at room temperature to obtain the modified fuchsin diluent b.
[0029] The preparation method of saturated picric acid aqueous solution is as follows: Under ventilated conditions at 20℃, 0.06 parts of picric acid crystals are first added to 1 part of distilled water and stirred at 150 rpm until completely dissolved. Using 1 part of distilled water as a standard, 0.03 parts of picric acid crystals are added and stirred at 150 rpm for 10 minutes to ensure that the picric acid crystals are completely dissolved and the solution is clear. This process is repeated. When solid residue is observed at the bottom of the container and no further dissolution occurs when stirring, it indicates that saturation has been reached. Stirring is stopped and the solution is allowed to stand for 2 hours. Then, the clear, bright yellow supernatant is filtered and transferred to a brown glass bottle. It is stored in a cool, dark place to obtain saturated picric acid aqueous solution.
[0030] An economical collagen fiber staining kit contains nine dropper bottles, each containing a different reagent: modified iron hematoxylin (prepared by mixing modified iron hematoxylin a solution and modified iron hematoxylin b solution in a 1:1 volume ratio), Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin solution (prepared by mixing modified fuchsin a solution and modified fuchsin dilution b solution in a 1:9 volume ratio), differentiation concentrate, acidic differentiation solution, bluing solution, and Scott's blueing solution.
[0031] Example 2: A method for preparing an economical collagen fiber staining reagent, comprising the following reagents: modified iron hematoxylin, Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin solution, differentiation concentrate, acidic differentiation solution, bluing solution, and Scott's blueing solution. Modified iron hematoxylin was prepared by mixing modified iron hematoxylin a solution and modified iron hematoxylin b solution in equal volumes at a ratio of 1:1. The modified fuchsin solution is prepared by mixing modified fuchsin dye solution a and modified fuchsin diluent b at a volume ratio of 1:9.
[0032] The modified iron hematoxylin a solution is prepared as follows: Hematoxylin colorant is added to anhydrous ethanol in a 35°C water bath and stirred continuously at 250 rpm until completely dissolved; then glycerol, ascorbic acid and polyethylene glycol 400 are added sequentially, and after each addition, the mixture is stirred continuously at 250 rpm for 7 minutes until homogeneous; after all components have been added, the mixed solution is transferred to a light-proof container and stirred at 250 rpm for 15 minutes at room temperature, then sealed and allowed to stand and mature at 4°C for 5 weeks in the dark to obtain the modified iron hematoxylin a solution.
[0033] The mass ratio of hematoxylin colorant to anhydrous ethanol is 1:80; the mass ratio of hematoxylin colorant to glycerol is 1:0.8; the mass ratio of hematoxylin colorant to ascorbic acid is 1:0.05; and the mass ratio of hematoxylin colorant to polyethylene glycol 400 is 1:2.5.
[0034] The modified iron hematoxylin b solution was prepared as follows: Under ventilated conditions and at 25°C, deionized water was added to a 30% ferric chloride solution and stirred at 200 rpm for 2 min. Then, while continuously stirring, concentrated hydrochloric acid was added dropwise at 1.5 mL / min, and the pH was controlled at 2.0. After the addition was complete, sodium citrate and potassium aluminum sulfate powder were added sequentially, and the stirring speed was increased to 250 rpm. The stirring was continued for 12 min until the solid was completely dissolved and the solution was clear. Finally, deionized water was added to bring the volume to the required total volume, and the solution was stirred at 150 rpm for 5 min to mix. The solution was then transferred to a brown bottle and stored at room temperature away from light to obtain the modified iron hematoxylin b solution.
[0035] The mass ratio of ferric chloride solution to deionized water is 1:0.65; the mass ratio of ferric chloride solution to concentrated hydrochloric acid is 1:0.22; the mass ratio of ferric chloride solution to sodium citrate is 1:0.06; and the mass ratio of ferric chloride solution to potassium aluminum sulfate powder is 1:0.03.
[0036] The modified eosin staining solution was prepared as follows: 97 parts of a pH 4.8 acetate-sodium acetate buffer solution and 1 part of water-soluble eosin Y powder were mixed at 25℃ and stirred at 250 rpm for 35 min until the powder was completely dissolved, forming a uniform red clear solution. 0.10 parts of polysorbate-20 and 0.5 parts of benzyl alcohol were added to the clear solution sequentially, and the stirring speed was reduced to 175 rpm. The mixture was stirred for 12 min to ensure that all added components were thoroughly mixed. Finally, the obtained staining solution was adjusted to the target volume with a pH 4.8 acetate-sodium acetate buffer solution and homogenized by stirring at 150 rpm for 5 min. The solution was then transferred to a light-protected reagent bottle and stored at room temperature to obtain the modified eosin staining solution.
[0037] The modified fuchsin dye solution a is prepared as follows: Under light-protected conditions at 25°C, 100 parts by mass of a 1% acetic acid aqueous solution are mixed with 1 part of acid fuchsin powder, and stirred continuously at 250 rpm for 55 min until the powder dissolves and the solution is uniformly deep red; then, 0.025 parts by mass of sodium benzoate and 0.10 parts by mass of polyethylene glycol 6000 are added sequentially, and the mixture is stirred continuously at 17 min to ensure that the added components are fully dispersed and dissolved; finally, the bottle wall is washed with a 1% acetic acid solution and the volume is adjusted to the target volume, and then stirred at 175 rpm for 7 min to make the system completely homogeneous. The solution is then transferred to a light-protected container, sealed, and stored at room temperature to obtain the modified fuchsin dye solution a.
[0038] The modified fuchsin diluent b was prepared as follows: At 25°C and under light-protected and ventilated conditions, 1 part of saturated picric acid aqueous solution was mixed with 0.12 parts of 70% ethanol solution, and stirred at 200 rpm for 7 minutes. While maintaining the stirring speed, 0.012 parts of glacial acetic acid solution was added to stabilize the pH at 2.2, followed by 0.008 parts of saturated sodium chloride aqueous solution. The stirring speed was increased to 250 rpm, and stirring continued for 17 minutes until all components were completely dissolved and the solution was clear. After mixing, the stirring speed was reduced to 125 rpm and stirred for 5 minutes to complete homogenization. The solution was then transferred to a light-protected reagent bottle, sealed, and stored at room temperature to obtain the modified fuchsin diluent b.
[0039] The preparation method of saturated picric acid aqueous solution is as follows: Under ventilated conditions at 25°C, 0.07 parts of picric acid crystals are first added to 1 part of distilled water and stirred at 200 rpm until completely dissolved. Using 1 part of distilled water as a standard, 0.04 parts of picric acid crystals are added and stirred at 200 rpm for 12 minutes to ensure that the picric acid crystals are completely dissolved and the solution is clear. This process is repeated. When solid residue is observed at the bottom of the container and no further dissolution occurs when stirring, it indicates that saturation has been reached. Stirring is stopped and the solution is allowed to stand for 3 hours. Then, the clear, bright yellow supernatant is filtered and transferred to a brown glass bottle. It is stored in a cool, dark place to obtain saturated picric acid aqueous solution.
[0040] An economical collagen fiber staining kit contains nine dropper bottles, each containing a different reagent: modified iron hematoxylin (prepared by mixing modified iron hematoxylin a solution and modified iron hematoxylin b solution in a 1:1 volume ratio), Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin solution (prepared by mixing modified fuchsin a solution and modified fuchsin dilution b solution in a 1:9 volume ratio), differentiation concentrate, acidic differentiation solution, bluing solution, and Scott's blueing solution.
[0041] Example 3: A method for preparing an economical collagen fiber staining reagent, comprising the following reagents: modified iron hematoxylin, Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin solution, differentiation concentrate, acidic differentiation solution, bluing solution, and Scott's blueing solution. Modified iron hematoxylin was prepared by mixing modified iron hematoxylin a solution and modified iron hematoxylin b solution in equal volumes at a ratio of 1:1. The modified fuchsin solution is prepared by mixing modified fuchsin dye solution a and modified fuchsin diluent b at a volume ratio of 1:9.
[0042] The modified iron hematoxylin a solution is prepared as follows: Hematoxylin colorant is added to anhydrous ethanol in a 45°C water bath and stirred continuously at 300 rpm until completely dissolved; then glycerol, ascorbic acid and polyethylene glycol 400 are added sequentially, and after each addition, the mixture is stirred continuously at 300 rpm for 10 minutes until homogeneous; after all components have been added, the mixed solution is transferred to a light-proof container and stirred at 300 rpm for 20 minutes at room temperature, then sealed and allowed to stand and mature at 4°C for 6 weeks in the dark to obtain the modified iron hematoxylin a solution.
[0043] The mass ratio of hematoxylin colorant to anhydrous ethanol is 1:85; the mass ratio of hematoxylin colorant to glycerol is 1:1; the mass ratio of hematoxylin colorant to ascorbic acid is 1:0.06; and the mass ratio of hematoxylin colorant to polyethylene glycol 400 is 1:3.
[0044] The modified iron hematoxylin b solution was prepared as follows: Under ventilated conditions and at 30°C, deionized water was added to a 30% ferric chloride solution and stirred at 250 rpm for 2 min. Then, concentrated hydrochloric acid was added dropwise at 2 mL / min while continuously stirring, and the pH value was controlled at 2.5. After the addition was completed, sodium citrate and potassium aluminum sulfate powder were added in sequence, and the stirring speed was increased to 300 rpm. The stirring was continued for 15 min until the solid was completely dissolved and the solution was clear. Finally, deionized water was added to make up the required total volume, and the solution was stirred at 200 rpm for 5 min to mix. The solution was then transferred to a brown bottle and stored at room temperature away from light to obtain the modified iron hematoxylin b solution.
[0045] The mass ratio of ferric chloride solution to deionized water is 1:0.7; the mass ratio of ferric chloride solution to concentrated hydrochloric acid is 1:0.25; the mass ratio of ferric chloride solution to sodium citrate is 1:0.07; and the mass ratio of ferric chloride solution to potassium aluminum sulfate powder is 1:0.04.
[0046] The modified eosin staining solution is prepared as follows: 100 parts of a pH 4.8 acetate-sodium acetate buffer solution and 1 part of water-soluble eosin Y powder are mixed at 30℃ and stirred at 300 rpm for 40 min until the powder is completely dissolved, forming a uniform red clear solution. 0.12 parts of polysorbate-20 and 0.6 parts of benzyl alcohol are added to the clear solution sequentially, and the stirring speed is reduced to 200 rpm and stirred for 15 min to ensure that all added components are thoroughly mixed. Finally, the obtained staining solution is adjusted to the target volume with a pH 4.8 acetate-sodium acetate buffer solution and homogenized by stirring at 200 rpm for 5 min. The solution is then transferred to a light-protected reagent bottle and stored at room temperature to obtain the modified eosin staining solution.
[0047] The modified fuchsin dye solution a is prepared as follows: Under light-protected conditions at 30°C, 105 parts by mass of a 1% acetic acid aqueous solution are mixed with 1 part of acid fuchsin powder, and stirred continuously at 300 rpm for 60 min until the powder dissolves and the solution is uniformly deep red; then, 0.03 parts by mass of sodium benzoate and 0.12 parts by mass of polyethylene glycol 6000 are added sequentially, and the mixture is stirred continuously at 20 min to ensure that the added components are fully dispersed and dissolved; finally, the bottle wall is washed with a 1% acetic acid solution and the volume is adjusted to the target volume, and then stirred at 200 rpm for 10 min to make the system completely homogeneous. The solution is then transferred to a light-protected container, sealed, and stored at room temperature to obtain the modified fuchsin dye solution a.
[0048] The modified fuchsin diluent b is prepared as follows: Under conditions of 30°C and protection from light and ventilation, 1 part of saturated picric acid aqueous solution and 0.15 parts of 70% ethanol solution are mixed and stirred at 250 rpm for 10 min. While maintaining the stirring speed, 0.015 parts of glacial acetic acid solution are added to control the pH to be stable at 2.5, and then 0.009 parts of saturated sodium chloride aqueous solution are added. The stirring speed is increased to 300 rpm and stirred for another 20 min until all components are completely dissolved and the solution is clear. After mixing, the stirring speed is reduced to 150 rpm and stirred for 5 min to complete homogenization. The solution is then transferred to a light-protected reagent bottle, sealed, and stored at room temperature to obtain the modified fuchsin diluent b.
[0049] The preparation method of saturated picric acid aqueous solution is as follows: Under 30℃ and ventilation conditions, 0.08 parts of picric acid crystals are first added to 1 part of distilled water and stirred at 250 rpm until completely dissolved. Using 1 part of distilled water as a standard, 0.05 parts of picric acid crystals are added and stirred at 250 rpm for 15 minutes to ensure that the picric acid crystals are completely dissolved and the solution is clear. This process is repeated. When solid residue is observed at the bottom of the container and no further dissolution occurs when stirring, it indicates that saturation has been reached. Stirring is stopped and the solution is allowed to stand for 4 hours. Then, the clear, bright yellow supernatant is filtered and transferred to a brown glass bottle. It is stored in a cool, dark place to obtain saturated picric acid aqueous solution.
[0050] An economical collagen fiber staining kit contains nine dropper bottles, each containing a different reagent: modified iron hematoxylin (prepared by mixing modified iron hematoxylin a solution and modified iron hematoxylin b solution in a 1:1 volume ratio), Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin solution (prepared by mixing modified fuchsin a solution and modified fuchsin dilution b solution in a 1:9 volume ratio), differentiation concentrate, acidic differentiation solution, bluing solution, and Scott's blueing solution.
[0051] Cole's hematoxylin solution is prepared from hematoxylin dye, potassium aluminum sulfate dodecahydrate, iodine and anhydrous ethanol; Sirius red staining solution is prepared by mixing Sirius red and a saturated picric acid aqueous solution; The differentiation concentrate was prepared by mixing glacial acetic acid and deionized water; The acidic differentiation solution is prepared from hydrochloric acid, anhydrous ethanol and deionized water; Blueing solution is made by dissolving lithium carbonate powder in deionized water; Scott's blueing solution is made by dissolving sodium bicarbonate powder and magnesium sulfate heptahydrate powder in deionized water.
[0052] Modified iron hematoxylin is designated as reagent A, Cole's hematoxylin solution as reagent B, Sirius red staining solution as reagent C, modified eosin staining solution as reagent D, modified fuchsin solution as reagent E, differentiation concentrate as reagent F, acidic differentiation solution as reagent G, blueing solution as reagent H, and Scott's blueing solution as reagent I.
[0053] Reagent combination instructions: 1. Reagents (A), (C), and (F) can form a Sirius red staining experimental series; 2. Reagents (B), (D), (G), and (H) can form a series of HE staining experiments; 3. Reagents (A), (E), and (G) can form a VG staining experimental series; 4. Reagent (A), reagent (G), and reagent (I) can be used to form Masson staining and nuclear staining. 5. Reagents (A) and (B) can be used to prepare a series of water-soluble nuclear stains and DAB alcohol-soluble counterstains for immunohistochemistry experiments; 6. Reagents (C), (D), and (E) can form a series of cell smear and staining experiments.
[0054] Comparative Example 1: The difference between this comparative example and Example 1 is that this comparative example uses unmodified iron hematoxylin prepared by conventional methods.
[0055] Comparative Example 2 differs from Example 1 in that it uses an unmodified eosin dye solution prepared using conventional methods.
[0056] Comparative Example 3 differs from Example 1 in that it uses unmodified fuchsin solution prepared by conventional methods.
[0057] Performance testing: The staining reagents prepared in Examples 1, 2, 3, Comparative Examples 1, 2, and 3 were subjected to performance testing. Reagent stability (effective shelf life) test: The reagents prepared according to the examples and comparative proportions were aliquoted and stored in a suitable storage environment for a long period of time. Samples were taken out at preset time points (e.g., 0, 1, 3, 6, 12 months), and the changes in their appearance (precipitate, color, clarity) and physicochemical indicators (e.g., pH value) were systematically detected. The staining effect of aged reagents and fresh reagents on the same batch of tissue sections was quantitatively compared through standardized staining experiments. By analyzing the trend of changes in various indicators over time, the effective shelf life of the reagents under the premise of maintaining qualified staining efficacy was determined, thereby verifying their storage stability. The test was conducted in accordance with the Guidelines for Drug and Reagent Stability Testing in the General Chapters of the Pharmacopoeia of the People's Republic of China. Staining uniformity (coefficient of variation CV%) test: First, standardized tissue sections were grouped and stained according to a uniform procedure. Then, under strictly consistent microscope optics and camera parameters, high-resolution digital images of collagen fiber-rich areas in each group of sections were acquired. Subsequently, using professional image analysis software, a large number of measurement points were systematically and uniformly distributed within the collagen target area of each image to obtain the optical density or grayscale value of each point. Finally, the coefficient of variation (CV%) of all measurements within the same image was calculated as an indicator to evaluate the staining uniformity. The lower the CV% value, the more uniform the color. The test is based on the image uniformity evaluation in ISO 19227:2018 "Microscopes — Digital imaging systems — Characteristics and methods of measurement". Color contrast test: After staining standardized sections containing clearly defined contrast tissues, digital images were acquired under strictly consistent imaging conditions. Specialized software was used to separate the color channels corresponding to the target dye, and collagen-rich areas (target areas) and adjacent non-collagen tissue areas (contrast areas) were precisely selected, and their average optical density values were measured. Finally, the absolute value of the difference between the two was calculated as the color contrast index (ΔOD). A larger ΔOD value indicates a sharper contrast. The test was conducted according to ISO 19227:2018 "Microscopes—Digital imaging systems—Characteristics and methods of measurement" for image uniformity evaluation.
[0058] The obtained test data are recorded in Table 1 below:
[0059] By comparing and analyzing the relevant data in Table 1, it can be seen that the staining reagent prepared by the present invention using an economical collagen fiber staining kit and its preparation method has a long shelf life, indicating its good stability; the coefficient of variation (CV%) is low, indicating uniform staining results; and the color contrast index (ΔOD) is high, indicating that the target and background are clearly distinguished after staining, resulting in high image clarity. Therefore, the economical collagen fiber staining kit and its preparation method provided by the present invention have a broader market prospect and are more suitable for promotion.
[0060] In the description of this specification, references to terms such as "an embodiment," "example," and "specific example" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.
[0061] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims
1. A method for preparing an economical collagen fiber dyeing reagent, characterized in that, The reagents include: modified iron hematoxylin, Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin solution, differentiation concentrate, acidic differentiation solution, blueing solution, and Scott's blueing solution. The modified iron hematoxylin is prepared by mixing modified iron hematoxylin a solution and modified iron hematoxylin b solution in equal volumes at a ratio of 1:
1. The modified fuchsin solution is prepared by mixing modified fuchsin dye solution a and modified fuchsin diluent b at a volume ratio of 1:
9.
2. The method for preparing an economical collagen fiber dyeing reagent according to claim 1, characterized in that, The modified iron hematoxylin a solution is prepared as follows: Hematoxylin colorant is added to anhydrous ethanol in a water bath at 25-45℃ and stirred continuously until completely dissolved; then glycerol, ascorbic acid and polyethylene glycol 400 are added sequentially, and stirring is continued for 5-10 minutes after each addition until the mixture is homogeneous; after all components have been added, the mixed solution is transferred to a light-proof container, stirred at room temperature, then sealed and allowed to stand and mature at 4℃ in the dark for 4-6 weeks to obtain the modified iron hematoxylin a solution.
3. The method for preparing an economical collagen fiber dyeing reagent according to claim 2, characterized in that: The mass ratio of hematoxylin colorant to anhydrous ethanol is 1:(75-85); the mass ratio of hematoxylin colorant to glycerol is 1:(0.6-1); the mass ratio of hematoxylin colorant to ascorbic acid is 1:(0.04-0.06); and the mass ratio of hematoxylin colorant to polyethylene glycol 400 is 1:(2-3).
4. The method for preparing an economical collagen fiber dyeing reagent according to claim 1, characterized in that, The modified iron hematoxylin b solution is prepared as follows: deionized water is added to a 30% ferric chloride solution and stirred until homogeneous. Then, concentrated hydrochloric acid is added dropwise while continuously stirring, and the pH value is controlled at 1.5-2.
5. After the addition is complete, sodium citrate and potassium aluminum sulfate powder are added in sequence, and the mixture is stirred continuously until the solid is completely dissolved and the solution is clear, thus obtaining the modified iron hematoxylin b solution.
5. The method for preparing an economical collagen fiber dyeing reagent according to claim 4, characterized in that: The mass ratio of ferric chloride solution to deionized water is 1:(0.6-0.7); the mass ratio of ferric chloride solution to concentrated hydrochloric acid is 1:(0.2-0.25); the mass ratio of ferric chloride solution to sodium citrate is 1:(0.05-0.07); and the mass ratio of ferric chloride solution to potassium aluminum sulfate powder is 1:(0.02-0.04).
6. The method for preparing an economical collagen fiber dyeing reagent according to claim 1, characterized in that, The modified eosin staining solution is prepared as follows: 95-100 parts of an acetate-sodium acetate buffer solution with a pH of 4.8 are mixed with 1 part of water-soluble eosin Y powder, and the mixture is continuously stirred to form a uniform red clear solution. 0.08-0.12 parts of polysorbate-20 and 0.4-0.6 parts of benzyl alcohol are added to the clear solution in sequence, and the mixture is continuously stirred until the added components are fully mixed. Finally, the solution is brought to a fixed volume and homogenized to obtain the modified eosin staining solution.
7. The method for preparing an economical collagen fiber dyeing reagent according to claim 1, characterized in that, The modified fuchsin dye solution a is prepared as follows: 95-105 parts of 1% acetic acid aqueous solution are mixed with 1 part of acid fuchsin powder and stirred continuously until the powder is dissolved and the solution is uniformly deep red; then 0.02-0.03 parts of sodium benzoate and 0.08-0.12 parts of polyethylene glycol 6000 are added sequentially and stirred continuously until the added components are evenly dispersed and dissolved to obtain the modified fuchsin dye solution a.
8. The method for preparing an economical collagen fiber dyeing reagent according to claim 1, characterized in that, The modified fuchsin diluent b is prepared as follows: 1 part of saturated picric acid aqueous solution is mixed with 0.10-0.15 parts of 70% ethanol solution and stirred until fully mixed; then, under stirring conditions, 0.010-0.015 parts of glacial acetic acid solution is added to control the pH to be stable at 2.0-2.5, and then 0.007-0.009 parts of saturated sodium chloride aqueous solution is added. The stirring speed is increased until all components are completely dissolved and the solution is clear; after mixing, the stirring speed is reduced and stirring is continued to complete homogenization, thus obtaining modified fuchsin diluent b.
9. The method for preparing an economical collagen fiber dyeing reagent according to claim 8, characterized in that, The method for preparing the saturated picric acid aqueous solution is as follows: First, add 0.06-0.08 parts of picric acid crystals to 1 part of distilled water and stir continuously until completely dissolved; then, based on 1 part of distilled water, add 0.03-0.05 parts of picric acid crystals and stir until the picric acid crystals are completely dissolved and the solution is clear. Repeat this process; when solid residue is observed at the bottom of the container and stirring no longer dissolves, it indicates that saturation has been reached; stop stirring and let stand, then filter and take the supernatant to obtain the saturated picric acid aqueous solution.
10. An economical collagen fiber staining kit, prepared by the method for preparing an economical collagen fiber staining reagent according to any one of claims 1-9, characterized in that: The kit contains 11 dropper bottles, each containing a different reagent. The reagents in the 11 dropper bottles are: modified iron hematoxylin a solution, modified iron hematoxylin b solution, Cole's hematoxylin solution, Sirius red staining solution, modified eosin staining solution, modified fuchsin staining solution a, modified fuchsin dilution solution b, differentiation concentrate, acidic differentiation solution, bluing solution, and Scott's blueing solution.