Improved masson three-color dyeing method

Abstract

The invention provides an improved masson three-color dyeing method. The method comprises the following steps: immersing a tissue slice into a Bouin solution for pretreatment mordanting; dropwise adding an iron hematoxylin staining solution on the tissue slice for staining; immersing the tissue slice into a Bobhlet red staining solution for staining; immersing the tissue slices into a phosphomolybdic acid aqueous solution for fiber differentiation; immersing the tissue slice into aniline blue staining fluid for staining; and sealing the slice to obtain the improved tissue slice. According to the method disclosed by the invention, a Bobillet scarlet staining solution is used for replacing a traditional richlet acid staining solution, and a Bouin solution is combined for pre-treating the section, so that the staining effect is improved, and the staining contrast of collagenous fibers and other tissues is more obvious.

Description

Technical Field

[0001] The invention belongs to the technical field of biology and pathology, and in particular relates to an improved Masson trichrome staining method, which makes the coloring effect comparison of each tissue in the staining result more obvious by replacing a specific staining reagent. Background Art

[0002] Masson's trichrome staining is one of the commonly used staining methods in histology and pathology, and is mainly used to distinguish tissue components such as collagen fibers and muscle fibers.

[0003] However, the traditional Masson trichrome staining method uses Ponceau acid staining solution for staining, and its staining effect may not be ideal in some cases, especially when it is necessary to show the contrast between the various tissue components more clearly.

[0004] Therefore, it is urgent to provide an improved Masson's trichrome staining method, thereby improving the staining effect and making the staining contrast between collagen fibers and other tissues more obvious. Summary of the invention

[0005] Based on the defects of the prior art, the purpose of the present invention is to provide an improved Masson's trichrome staining method, which uses Biblical scarlet staining solution to replace the traditional Ponceau acid staining solution, and combines Bouin's solution to pre-treat the sections, thereby improving the staining effect and making the staining contrast between collagen fibers and other tissues more obvious.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] In one aspect, the present invention provides a modified Masson's trichrome staining method, the method comprising the following steps:

[0008] Step 1, immersing the tissue sections in Bouin's solution for pre-treatment and mordant dyeing;

[0009] Step 2, then dripping iron hematoxylin staining solution on the tissue section for staining;

[0010] Step 3, immersing the tissue sections in Biebrich scarlet staining solution for staining;

[0011] Step 4, immersing the tissue slices in a phosphomolybdic acid aqueous solution for fiber differentiation;

[0012] Step 5, immersing the tissue section in aniline blue staining solution for staining;

[0013] Improved tissue sections were obtained after sealing.

[0014] In the above staining method, preferably, in step 1, the preparation method of the Bouin's solution is: 70 ml of saturated picric acid solution, 25 ml of formalin (4% formaldehyde aqueous solution) and 5 ml of glacial acetic acid are mixed uniformly to prepare Bouin's solution.

[0015] In the above-mentioned staining method, preferably, the pre-treatment temperature in Bouin's solution is 35 to 37° C., and the treatment time is 30 to 80 min.

[0016] In the above dyeing method, preferably, the step of washing the tissue sections is further included after the mordant treatment, specifically by washing with running water until the yellow color on the tissue sections disappears.

[0017] In the above staining method, preferably, in step 2, the preparation method of the iron hematoxylin staining solution is:

[0018] Liquid A: Add 1g of hematoxylin powder to 100ml of anhydrous ethanol and heat to completely dissolve the hematoxylin in the anhydrous ethanol to obtain Liquid A;

[0019] Solution B: Add 4 mL of 30% anhydrous ferric chloride solution and 1 mL of concentrated hydrochloric acid into 100 mL of deionized water and mix well to obtain Solution B;

[0020] Solution A and solution B are mixed to obtain the iron hematoxylin staining solution.

[0021] In the above staining method, preferably, in step three, the staining time using iron hematoxylin staining solution is 5 minutes.

[0022] In the above staining method, preferably, the step of differentiating and anti-blueing the tissue sections is further included after the iron hematoxylin staining treatment.

[0023] In the above staining method, preferably, the differentiation is performed by immersing the tissue sections in 0.5% hydrochloric acid ethanol solution for 6 seconds.

[0024] In the above staining method, preferably, the anti-blueing is performed by washing the tissue sections with running water for 5 minutes.

[0025] In the above staining method, preferably, in step 3, the preparation method of the Biebrich scarlet staining solution is:

[0026] Take 1 g of Biebrich's scarlet powder and add it to 100 ml of deionized water until the powder is completely dissolved to prepare Biebrich's scarlet staining solution.

[0027] In the above staining method, preferably, the staining time using Biebrich scarlet staining solution is 5 minutes.

[0028] In the above staining method, preferably, the step of washing the tissue slices is further included after the Biebrich scarlet staining treatment; specifically, deionized water is used to wash the dye on the surface of the tissue slices, and absorbent paper is used to absorb water droplets on the surface of the tissue slices.

[0029] In the above dyeing method, preferably, in step 4, the preparation method of the phosphomolybdic acid aqueous solution is: take 1g of phosphomolybdic acid powder and add it to 100ml of deionized water until the powder is completely dissolved to prepare the phosphomolybdic acid aqueous solution.

[0030] In the above staining method, preferably, the immersion time of the tissue sections in the phosphomolybdic acid aqueous solution for fiber differentiation is 1 to 3 minutes; and the fiber differentiation is observed under a microscope.

[0031] In the above dyeing method, preferably, in step five, the preparation method of the aniline blue dyeing solution is: take 0.5 g of aniline blue powder and add it into 100 ml of deionized water until the powder is completely dissolved to prepare the aniline blue dyeing solution.

[0032] In the above staining method, preferably, the time for staining the tissue section with aniline blue staining solution is 3 minutes.

[0033] In the above staining method, preferably, the step of washing and dehydrating the tissue sections is further included after the aniline blue staining treatment.

[0034] In the above staining method, preferably, the washing is performed by immersing the tissue section in a 1% acetic acid aqueous solution for 1 min.

[0035] In the above staining method, preferably, the dehydration is to sequentially soak the tissue sections in anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, anhydrous ethanol for 5 minutes, and an environmentally friendly transparent agent for 5 minutes.

[0036] In the above staining method, preferably, the tissue sections further include the steps of solidifying, dewaxing, hydrating and washing the tissue sections in sequence before mordanting.

[0037] In the above staining method, preferably, the curing is to bake the tissue sections in a 60° C. thermostat for 60 min.

[0038] In the above staining method, preferably, the dewaxing is to immerse the tissue sections in xylene for 10 minutes, and repeat once.

[0039] In the above staining method, preferably, the hydration is to soak the tissue sections in anhydrous ethanol for 2 minutes, replace with anhydrous ethanol for 2 minutes, soak in 95% ethanol for 2 minutes, and soak in 80% ethanol for 2 minutes.

[0040] In the above staining method, preferably, the washing is to rinse the tissue sections with running water for 5 minutes.

[0041] In the above staining method, preferably, the sealing is performed by using environmentally friendly transparent adhesive to seal the tissue sections after the staining process.

[0042] On the other hand, the present invention also provides an improved tissue section, which is prepared by the above-mentioned staining method.

[0043] Beneficial effects of the present invention:

[0044] The method of the present invention uses Biebrich scarlet staining solution to replace the traditional Ponceau acid staining solution, and combines Bouin's solution to pre-treat the slice, thereby improving the staining effect and making the staining contrast between collagen fibers and other tissues more obvious.

[0045] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 The staining diagram of the epidermis and fibrous connective tissue by the improved Masson trichrome staining method of Example 1 of the present invention;

[0048] Figure 2 This is the staining diagram of the epidermis and fibrous connective tissue using the conventional Masson trichrome staining method of Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention does not particularly limit the contents.

[0050] Preparation of experimental reagents:

[0051] (1) Bouin's solution: 70 ml of saturated picric acid solution, 25 ml of formalin (4% formaldehyde aqueous solution), and 5 ml of glacial acetic acid.

[0052] (2) Iron hematoxylin red staining solution: Solution A: Add 1g of hematoxylin powder to 100ml of anhydrous ethanol and heat it slightly to completely dissolve the hematoxylin in the anhydrous ethanol. Solution B: Add 4ml of 30% anhydrous ferric chloride solution and 1ml of concentrated hydrochloric acid to 100ml of deionized water and mix well. When using, mix solution A and solution B in equal proportions. The validity period is 1 day.

[0053] (3) Hydrochloric acid ethanol: Take 0.5 ml of concentrated hydrochloric acid and add it to 99.5 ml of anhydrous ethanol.

[0054] (4) Biebrich's scarlet staining solution: Take 1 g of Biebrich's scarlet powder and add it to 100 ml of deionized water until the powder is completely dissolved.

[0055] (5) Aniline blue staining solution: Take 0.5 g of aniline blue powder and add it into 100 ml of deionized water until the powder is completely dissolved.

[0056] (6) Acetic acid aqueous solution: add 1 ml of glacial acetic acid to 99 ml of deionized water.

[0057] (7) Phosphomolybdic acid aqueous solution: 1 g of phosphomolybdic acid powder was added into 100 ml of deionized water until the powder was completely dissolved.

[0058] Embodiment 1:

[0059] This embodiment provides a modified Masson trichrome staining method for pig skin tissue, the method comprising the following steps:

[0060] (1) Curing: Bake the tissue sections in a 60°C thermostat for 60 min;

[0061] (2) Dewaxing: Soak tissue sections in xylene for 10 min, then replace xylene and soak for another 10 min;

[0062] (3) Hydration: Soak the tissue sections in anhydrous ethanol for 2 min, then in anhydrous ethanol for 2 min, then in 95% ethanol for 2 min, and finally in 80% ethanol for 2 min.

[0063] (4) Washing: Rinse tissue sections with running water for 2 min;

[0064] (5) Mordanting: Place the tissue sections in Bouin's solution and heat in a 37°C oven for 1 h;

[0065] (6) Washing: Rinse with running water until the yellow color on the tissue section disappears;

[0066] (7) Iron hematoxylin staining: Add iron hematoxylin staining solution to the tissue section and stain for 5 minutes;

[0067] (8) Differentiation: The tissue sections were differentiated with 0.5% hydrochloric acid ethanol for 6 seconds;

[0068] (9) Anti-blueing: Rinse tissue sections with running water for 5 min;

[0069] (10) Bieblich scarlet staining: Immerse the tissue sections in Bieblich scarlet staining solution for 5 min;

[0070] (11) Washing: Use deionized water to wash away the stain on the surface of the tissue sections, and use absorbent paper to absorb the water droplets on the surface of the sections;

[0071] (12) Differentiation: Immerse the tissue sections in phosphomolybdic acid differentiation solution for 1-3 min and observe the fiber differentiation under a microscope;

[0072] (13) Aniline blue staining: After removing the tissue sections from the phosphomolybdic acid, immerse them directly in aniline blue staining solution for 3 min;

[0073] (14) Washing: Immerse the tissue sections in 1% acetic acid aqueous solution for 1 min;

[0074] (15) Dehydration: Soak the tissue sections in anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, xylene for 5 minutes, and xylene for 5 minutes.

[0075] (16) Sealing: Finally, use environmentally friendly transparent tape to seal the tissue sections.

[0076] Comparative Example 1:

[0077] This comparative example provides a conventional Masson staining method for pig skin tissue, which comprises the following steps:

[0078] (1) Dewaxing: Soak tissue sections in xylene for 10 min, then replace xylene and soak for another 10 min;

[0079] (2) Hydration: Soak in anhydrous ethanol for 2 min, replace with anhydrous ethanol for 2 min, soak in 95% ethanol for 2 min, and soak in 80% ethanol for 2 min;

[0080] (3) Washing: Rinse in running water for 2 minutes;

[0081] (4) Iron hematoxylin staining: add iron hematoxylin staining solution for 5 minutes;

[0082] (5) Differentiation: 0.5% hydrochloric acid ethanol differentiation for 6 seconds;

[0083] (6) Anti-blue: rinse with running water for 5 minutes;

[0084] (7) Use Ponceau acid red solution for 5 minutes;

[0085] (8) Soak in 2% glacial acetic acid solution for a while;

[0086] (9) Differentiation with 1% phosphomolybdic acid aqueous solution for 3 min;

[0087] (10) Dye with aniline blue for 5 min without washing;

[0088] (11) Soak in 0.2% glacial acetic acid aqueous solution for a while;

[0089] (12) 95% alcohol, anhydrous alcohol, xylene for transparency, and neutral gum for sealing.

[0090] The results of pathological histology experiments are shown in Table 1. Figure 1 and Figure 2 As shown, Figure 1 The staining diagram of the epidermis and fibrous connective tissue by the improved Masson trichrome staining method of Example 1 of the present invention; Figure 2 This is the staining diagram of the epidermis and fibrous connective tissue using the conventional Masson trichrome staining method of Comparative Example 1 of the present invention.

[0091] Table 1:

[0092]

[0093]

[0094] It can be seen from the above comparative experimental results that the sections stained by the modified Masson staining are more vividly stained than those stained by the conventional Masson staining, and the coloring contrast between the connective tissue and the epidermis is more obvious.

[0095] The present invention uses specific embodiments to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A modified Masson's trichrome staining method, characterized in that: The method comprises the following steps: Step 1, immersing the tissue sections in Bouin's solution for pre-treatment and mordant dyeing; Step 2, then dripping iron hematoxylin staining solution on the tissue section for staining; Step 3, immersing the tissue sections in Biebrich scarlet staining solution for staining; Step 4, immersing the tissue slices in a phosphomolybdic acid aqueous solution for fiber differentiation; Step 5, immersing the tissue section in aniline blue staining solution for staining; Improved tissue sections were obtained after sealing.

2. The dyeing method according to claim 1, characterized in that: In step 1, the preparation method of the Bouin solution is as follows: 70 ml of saturated picric acid solution, 25 ml of formalin (4% formaldehyde aqueous solution) and 5 ml of glacial acetic acid are mixed uniformly to prepare the Bouin solution; Preferably, the pretreatment temperature in Bouin's solution is 35-37°C and the treatment time is 30-80 min; Preferably, after the mordant treatment, the step of washing the tissue sections is further included, specifically by washing with running water until the yellow color on the tissue sections disappears.

3. The dyeing method according to claim 1, characterized in that: In step 2, the preparation method of the iron hematoxylin staining solution is: Liquid A: Add 1g of hematoxylin powder to 100ml of anhydrous ethanol and heat to completely dissolve the hematoxylin in the anhydrous ethanol to obtain Liquid A; Solution B: Add 4 mL of 30% anhydrous ferric chloride solution and 1 mL of concentrated hydrochloric acid into 100 mL of deionized water and mix well to obtain Solution B; Solution A and solution B are mixed to obtain the iron hematoxylin staining solution.

4. The dyeing method according to claim 3, characterized in that: In step 3, the staining time using iron hematoxylin staining solution is 5 minutes; Preferably, the method further comprises the steps of differentiating and anti-blueing the tissue sections after the iron hematoxylin staining treatment; The differentiation is performed by immersing the tissue slices in a 0.5% hydrochloric acid ethanol solution for 6 seconds; The anti-blueing is performed by washing the tissue sections with running water for 5 minutes.

5. The dyeing method according to claim 1, characterized in that: In step 3, the preparation method of the Biebrich scarlet staining solution is: Take 1 g of Biebrich's scarlet powder and add it to 100 ml of deionized water until the powder is completely dissolved to prepare Biebrich's scarlet staining solution; Preferably, the staining time using Biebrich scarlet staining solution is 5 minutes; Preferably, the step of washing the tissue slices is further included after the Biebrich scarlet staining treatment; specifically, the step of washing the stain on the surface of the tissue slices with deionized water and absorbing the water droplets on the surface of the tissue slices with absorbent paper.

6. The dyeing method according to claim 1, characterized in that: In step 4, the preparation method of the phosphomolybdic acid aqueous solution is: taking 1g of phosphomolybdic acid powder and adding it into 100ml of deionized water until the powder is completely dissolved, so as to prepare the phosphomolybdic acid aqueous solution; Preferably, the immersion time of the tissue sections in the phosphomolybdic acid aqueous solution for fiber differentiation is 1 to 3 minutes; and the fiber differentiation is observed under a microscope.

7. The dyeing method according to claim 1, characterized in that: In step 5, the preparation method of the aniline blue dyeing solution is: taking 0.5 g of aniline blue powder and adding it into 100 ml of deionized water until the powder is completely dissolved, so as to prepare the aniline blue dyeing solution; Preferably, the time for staining the tissue sections with aniline blue staining solution is 3 minutes; Preferably, the step of washing and dehydrating the tissue sections is further included after the aniline blue staining treatment; The washing step is to immerse the tissue sections in a 1% acetic acid aqueous solution for 1 minute; The dehydration step is to sequentially soak the tissue sections in anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, anhydrous ethanol for 30 seconds, xylene for 5 minutes, and xylene for 5 minutes.

8. The dyeing method according to claim 1, characterized in that: The tissue sections also include the steps of sequentially solidifying, dewaxing, hydrating and washing the tissue sections before mordanting; Preferably, the curing is to bake the tissue slices in a 60°C constant temperature oven for 60 minutes; Preferably, the dewaxing is performed by soaking the tissue sections in xylene for 10 min, and repeating the process once; Preferably, the hydration is to soak the tissue sections in anhydrous ethanol for 2 minutes, replace with anhydrous ethanol for 2 minutes, soak in 95% ethanol for 2 minutes, and soak in 80% ethanol for 2 minutes; Preferably, the washing is to rinse the tissue sections with running water for 5 minutes.

9. The dyeing method according to claim 1, characterized in that: The sealing is performed by using environmentally friendly transparent glue to seal the tissue sections after the staining process.

10. An improved tissue section, characterized in that: The improved tissue section is prepared by the staining method according to any one of claims 1 to 9.

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