Improved sarranine O fast green aniline blue dyeing method

Abstract

The invention provides an improved sarranine O fast green aniline blue dyeing method. The method comprises the following steps: dropwise adding an iron hematoxylin staining solution on a tissue slice for staining; immersing the tissue slices into fast green aniline blue staining fluid for staining; immersing the tissue slice into a sarranine O staining solution for staining; and sealing the slice to obtain the improved tissue slice. According to the method, improved sarranine O fast green aniline blue dyeing is used for replacing traditional single fast green dyeing liquid, hematoxylin dyeing is carried out during section dewaxing, and therefore the dyeing effect is improved, and compared with the single fast green dyeing liquid, the dyeing effect on bone tissue is better, and compared with cartilage tissue sarranine O dyeing, the dyeing method is more obvious.

Description

Technical Field

[0001] The invention belongs to the technical field of biology and pathology, and particularly relates to an improved safranin O fast green aniline blue staining method. The method replaces a single fast green staining solution in a conventional method. Compared with a single fast green staining solution, the improved staining method has a better coloring effect on bone tissue and is more obvious compared with safranin O staining of cartilage tissue. Background Art

[0002] In histological and pathological studies, the choice of staining method is of vital importance for the visualization of cell structures. Traditional staining techniques such as hematoxylin-eosin (H&E) staining are currently the most widely used routine staining methods to distinguish cell nuclei from cytoplasm. However, when different types of tissue components need to be distinguished in more detail, such as mineral deposition in bone tissue or matrix components in cartilage tissue, traditional staining methods may not provide sufficient contrast and specificity.

[0003] Fast green staining is a commonly used method for staining bone tissue, which can make the bone matrix appear green, thus facilitating the visualization of bone structure. However, a single Fast Green stain may not be sufficient to provide adequate contrast in some cases, especially in samples where bone tissue and other tissue types (such as cartilage) need to be clearly displayed at the same time. In addition, since Fast Green staining does not stain cartilage tissue well, it is difficult to obtain good contrast between the two tissue types in the same sample.

[0004] Therefore, it is urgent to provide an improved safranin O fast green aniline blue staining method, so as to improve the staining effect and achieve a better bone tissue coloring effect. Summary of the invention

[0005] Based on the defects of the prior art, the first purpose of the present invention is to provide an improved Safranin O fast green aniline blue staining method, which replaces the single fast green staining solution in the conventional method. The improved staining method has a better coloring effect on bone tissue than the single fast green staining solution, and is more obvious compared with the Safranin O staining of cartilage tissue; the second purpose of the present invention is to provide bone tissue sections dyed by the improved Safranin O fast green aniline blue staining method.

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

[0007] In one aspect, the present invention provides an improved method for dyeing safranin O fast green aniline blue, the method comprising the following steps:

[0008] Step 1, adding iron hematoxylin staining solution to the tissue section for staining;

[0009] Step 2, then immersing the tissue section in a fast green aniline blue staining solution for staining;

[0010] Step 3, then immersing the tissue section in Safranin O staining solution for staining;

[0011] Improved tissue sections were obtained after sealing.

[0012] In the above staining method, preferably, in step 1, the preparation method of the iron hematoxylin staining solution is:

[0013] 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;

[0014] 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;

[0015] Solution A and solution B are mixed to obtain the iron hematoxylin staining solution.

[0016] In the above-mentioned staining method, preferably, in step 1, the staining time using the iron hematoxylin staining solution is 5 minutes.

[0017] In the above staining method, preferably, in step 1, after the iron hematoxylin staining treatment, the step of differentiating and blueing the tissue sections is also included;

[0018] The differentiation is performed by immersing the tissue slices in a 0.5% hydrochloric acid ethanol solution for 6 seconds;

[0019] The anti-blueing is performed by washing the tissue sections with running water for 5 minutes.

[0020] In the above-mentioned dyeing method, preferably, in step 2, the preparation method of the fast green aniline blue dyeing solution is:

[0021] Solution A: Add 0.5 g of Fast Green powder into 100 ml of deionized water until the powder is completely dissolved to obtain Solution A;

[0022] Solution B: Add 1g of aniline blue powder to 1000ml of deionized water until the powder is completely dissolved to obtain Solution B;

[0023] A solution and B solution are mixed to obtain the fast green aniline blue dye solution.

[0024] In the above dyeing method, preferably, in step 2, the dyeing time of using the fast green aniline blue dyeing solution is 1 to 2 minutes.

[0025] In the above staining method, preferably, in step 2, the step of washing the tissue sections after the fast green aniline blue staining treatment is further included; specifically, the sections are immersed in a 1% acetic acid aqueous solution for 1 minute.

[0026] In the above-mentioned dyeing method, preferably, in step three, the preparation method of the Safranin O dyeing solution is: take 1g of Safranin O powder and add it into 1000mL of deionized water until the powder is completely dissolved.

[0027] In the above staining method, preferably, in step three, the staining time using Safranin O staining solution is 5 minutes.

[0028] In the above staining method, preferably, the step of washing and dehydrating the tissue sections is further included after the Safranin O staining treatment;

[0029] The washing is to rinse the tissue slices with water to clean the stain on the slice surface;

[0030] 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, an environmentally friendly transparent agent for 5 minutes, and an environmentally friendly transparent agent for 5 minutes.

[0031] In the above staining method, preferably, the tissue section further comprises the steps of solidifying, dewaxing, hydrating and washing the tissue section in sequence during the iron hematoxylin staining.

[0032] In the above staining method, preferably, the curing is to bake the tissue sections in a 60° C. thermostat for 60 min.

[0033] In the above staining method, preferably, the dewaxing is to immerse the tissue sections in xylene for 10 minutes, and repeat once.

[0034] 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.

[0035] In the above staining method, preferably, the washing is to rinse the tissue sections with running water for 5 minutes.

[0036] 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.

[0037] On the other hand, the present invention also provides an improved bone tissue slice, which is prepared by the above-mentioned staining method.

[0038] Beneficial effects of the present invention:

[0039] The method of the present invention replaces the single fast green staining solution in the conventional method. The improved staining method has a better coloring effect on bone tissue than the single fast green staining solution, and is more obvious compared with the cartilage tissue Safranin O staining.

[0040] 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

[0041] 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.

[0042] Figure 1 The staining diagram of pathological bone tissue and cartilage matrix of the improved safranin O fast green aniline blue staining method of Example 1 of the present invention;

[0043] Figure 2 This is a staining diagram of pathological bone tissue and cartilage matrix using the conventional Safranin O Fast Green staining method in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0044] 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.

[0045] Preparation of experimental reagents:

[0046] (1) Safranin O staining solution: Add 1 g of Safranin O powder into 1000 mL of deionized water until the powder is completely dissolved.

[0047] (2) Fast green aniline blue dyeing solution: Solution A: Add 0.5g of fast green powder to 1000ml of deionized water until the powder is completely dissolved. Solution B: Add 1g of aniline blue powder to 1000ml of deionized water until the powder is completely dissolved. When using, mix solution A and solution B in equal proportions.

[0048] (3) 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.

[0049] (4) Hydrochloric acid ethanol: Take 0.5 ml of concentrated hydrochloric acid and add it to 99.5 ml of anhydrous ethanol.

[0050] (5) Acetic acid aqueous solution: add 1 ml of glacial acetic acid to 99 ml of deionized water.

[0051] Embodiment 1:

[0052] This embodiment provides an improved safranin O fast green aniline blue staining method for rabbit knee joint tissue, the method comprising the following steps:

[0053] (1) Curing: Bake the tissue sections in a 60°C thermostat for 60 min;

[0054] (2) Dewaxing: Soak tissue sections in xylene for 10 min, then replace xylene and soak for another 10 min;

[0055] (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.

[0056] (4) Washing: Rinse tissue sections with running water for 2 min;

[0057] (5) Iron hematoxylin staining: Add iron hematoxylin staining solution to the tissue section and stain for 5 minutes;

[0058] (6) Differentiation: The tissue sections were differentiated with 0.5% hydrochloric acid ethanol for 6 seconds;

[0059] (7) Anti-blueing: Rinse tissue sections with running water for 5 min;

[0060] (8) Fast green aniline blue staining: Immerse the tissue sections in fast green aniline blue staining solution for 2 min;

[0061] (9) Washing: Immerse the sections in 1% acetic acid aqueous solution for 1 min;

[0062] (10) Safranin O staining: Immerse the sections in Safranin O staining solution for 5 min;

[0063] (11) Washing: Rinse the slices with water to clean the surface dye;

[0064] (12) 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, environmentally friendly transparent agent for 5 minutes, and environmentally friendly transparent agent for 5 minutes.

[0065] (13) Sealing: Finally, use environmentally friendly transparent glue to seal the tissue sections.

[0066] Comparative Example 1:

[0067] This comparative example provides a conventional safranin O fast green staining method for rabbit knee joint tissue, the method comprising the following steps:

[0068] (1) Dewaxing: Soak tissue sections in xylene for 10 min, then replace xylene and soak for another 10 min;

[0069] (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;

[0070] (3) Washing: Rinse in running water for 2 minutes;

[0071] (4) Iron hematoxylin staining: add iron hematoxylin staining solution for 5 minutes;

[0072] (5) Differentiation: 0.5% hydrochloric acid ethanol differentiation for 6 seconds;

[0073] (6) Anti-blue: rinse with running water for 5 minutes;

[0074] (7) Dye with fast green dye for 4 min;

[0075] (8) 1% glacial acetic acid (weak acid solution) for 15 seconds;

[0076] (9) Slice and dry;

[0077] (10) Staining with safranin solution for 3 min;

[0078] (11) 95% alcohol, anhydrous alcohol, xylene for transparency, and neutral gum for sealing.

[0079] The results of pathological histology experiments are shown in Table 1. Figure 1 and Figure 2 As shown, Figure 1 The image of pathological bone tissue and cartilage matrix obtained by the improved safranin O fast green aniline blue staining method of Example 1; Figure 2 This is a picture of pathological bone tissue and cartilage matrix obtained by conventional safranin O fast green staining method in comparative example 1.

[0080] Table 1:

[0081]

[0082] It can be seen from the above comparative experimental results that the sections stained with the improved Safranin O Fast Green are more vividly stained than the sections stained with the conventional Safranin O Fast Green, and the coloring contrast between the bone tissue and the cartilage matrix is ​​more obvious.

[0083] 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. An improved method for dyeing safranin O fast green aniline blue, characterized in that: The method comprises the following steps: Step 1, adding iron hematoxylin staining solution to the tissue section for staining; Step 2, then immersing the tissue section in a fast green aniline blue staining solution for staining; Step 3, then immersing the tissue section in Safranin O 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 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.

3. The dyeing method according to claim 1 or 2, characterized in that: In step 1, 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.

4. The dyeing method according to claim 1, characterized in that: In step 2, the preparation method of the fast green aniline blue dyeing solution is: Solution A: Add 0.5 g of Fast Green powder into 100 ml of deionized water until the powder is completely dissolved to obtain Solution A; Solution B: Add 1g of aniline blue powder to 1000ml of deionized water until the powder is completely dissolved to obtain Solution B; A solution and B solution are mixed to obtain the fast green aniline blue dye solution.

5. The dyeing method according to claim 1 or 4, characterized in that: In step 2, the fast green aniline blue dye solution is used for dyeing for 1 to 2 minutes; Preferably, the method further comprises washing the tissue sections after the fast green aniline blue staining treatment; specifically, the sections are immersed in a 1% acetic acid aqueous solution for 1 minute.

6. The dyeing method according to claim 1, characterized in that: In step three, the preparation method of the Safranin O dye solution is: take 1g of Safranin O powder and add it into 1000mL of deionized water until the powder is completely dissolved.

7. The dyeing method according to claim 1 or 6, characterized in that: In step 3, the staining time using Safranin O staining solution is 5 minutes; Preferably, the method further comprises washing and dehydrating the tissue sections after the Safranin O staining treatment; The washing is to rinse the tissue slices with water to clean the stain on the slice surface; 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, an environmentally friendly transparent agent for 5 minutes, and an environmentally friendly transparent agent for 5 minutes.

8. The dyeing method according to claim 1, characterized in that: The tissue section further comprises the steps of sequentially solidifying, dewaxing, hydrating and washing the tissue section during iron hematoxylin staining; 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 bone tissue slice, characterized in that: The improved bone tissue slice is prepared by the staining method according to any one of claims 1 to 9.

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