Dyeing agent and dyeing method for cross grains of rhabdoids

Abstract

The invention provides a rhabdoid cross grain staining agent and a staining method. The staining method comprises the following steps: S1, preparing a slide sample of fresh muscle tissue; s2, periodic acid oxidation washing; s3, dyeing with a rhabdoid cross grain dyeing agent, wherein the rhabdoid cross grain dyeing agent comprises the following components: a color-changing green 2R dyeing solution, methyl green, water and glacial acetic acid; s4, after dyeing, washing away redundant dye liquor with running water; s5, dropwise adding aniline blue dye liquor for re-dyeing; s6, washing off excessive dye liquor; the rhabdoid cross grain dyeing agent comprises the following components: a color-changing green 2R dyeing solution, methyl green, water and glacial acetic acid; the mass ratio of the color-changing green 2R staining solution to the methyl green is (3.6-4): 1; the volume ratio of the water to the glacial acetic acid is (98-99): (1-2); the invention provides a rhabdoid cross grain dyeing agent and a dyeing method, which can quickly and clearly judge whether cross grains are complete and whether tissues are fresh on fresh tissues by using two colors.

Description

Technical Field

[0001] The invention relates to a striated muscle striation staining agent and a staining method, belonging to the technical field of specimen staining. Background Art

[0002] Muscle tissue is composed of muscle cells and muscle fibers, and can be divided into skeletal muscle, cardiac muscle and smooth muscle. Skeletal muscle contains striations, and muscle fibers are connected and surrounded by connective tissue. The fibers are arranged in bundles, and each muscle fiber contains reticular fibers and a small amount of connective tissue. Myocardial fibers also contain striations, and the fibers are connected to each other to form a reticular structure. The outer surface of smooth muscle fibers contains reticular fibers and collagen fibers. According to the needs, different special staining and histochemical methods can be used to show the changes in components such as muscle fibers and connective tissue fibers.

[0003] Muscle tissue is composed of muscle cells and muscle fibers, with a striated structure. Mallory's phosphotungstic acid-hematoxylin-eosin staining method (PTAH) is usually used to show the changes in striated muscle. Mallory's phosphotungstic acid-hematoxylin-eosin staining method uses dyes in paraffin tissue sections. The dye can firmly bind to the selected tissue components and appear blue or brownish red. Mallory's phosphotungstic acid-hematoxylin-eosin staining solution: 0-1g hematoxylin, 2g phosphotungstic acid, 100ml distilled water. Heat hematoxylin in 20ml distilled water to dissolve, and then dissolve phosphotungstic acid in 80ml distilled water. After the hematoxylin is cooled, add the phosphotungstic acid aqueous solution, mix and mature at room temperature for several weeks to several months, and it can be stored for a long time. If it is urgently needed, 0.177g potassium permanganate can be added to promote its maturation.

[0004] The Mallory phosphotungstic acid-hematoxylin-eosin staining (PTAH) commonly used in the prior art has at least the following defects: paraffin section tissue needs to be dehydrated and waxed for more than ten hours, and the formaldehyde fixative, ethanol dehydrating agent, and xylene transparent agent used are all toxic or flammable substances. The conventional judgment of whether the horizontal stripes are complete is to perform hematoxylin staining or MASSON special staining on the basis of paraffin, which is not only cumbersome, but also requires professional personnel to perform sectioning before the experiment can be carried out. Figure 1-2 As shown, the PTAH method determines that the horizontal stripes are the same color, one light and one dark, which is not conducive to rapid and detailed distinction. Summary of the invention

[0005] The invention provides a striated muscle striated staining agent and a staining method which can quickly use two colors on fresh tissue to clearly judge whether the striated lines are complete and whether the tissue is fresh.

[0006] To achieve this object, the present invention provides the following technical solutions:

[0007] A first aspect of the present invention provides a method for staining striated muscle striations, comprising the following steps:

[0008] S1. Prepare slide samples of fresh muscle tissue;

[0009] S2, periodic acid oxidation washing;

[0010] S3, staining with a striated muscle stain, wherein the striated muscle stain comprises the following components: color-changing green 2R staining solution, methyl green, water, and glacial acetic acid;

[0011] S4, after dyeing, rinse off the excess dye with running water;

[0012] S5, adding aniline blue dye solution for counter-dyeing;

[0013] S6. Wash off excess dye.

[0014] In the present invention, a glass slide sample of fresh muscle tissue is used for staining, the glass slide preparation is simple, and the cumbersome paraffin sectioning step is avoided.

[0015] In the present invention, glacial acetic acid plays a dyeing promoting role, the color-changing green 2R dye solution combines with methyl green to react specifically with the fresh striated muscle color band to show red, the striated muscle tissue is stained with aniline blue to be blue-green, and the remaining tissues such as loose connective tissue between muscles are light green. The dyeing method of the present invention can show the stripes clearly in red and blue. Compared with the dyeing of striated muscle in the prior art, which can only distinguish the stripes from other tissues with the same color depth, the dyeing method of the present invention is more conducive to the detailed study of striated muscle, and provides more clear evidence for some scientific studies such as pharmacological reactions on muscle tissue.

[0016] Preferably, water includes but is not limited to distilled water, sterile water, and deionized water.

[0017] Preferably, in step S1, the method for preparing the glass slide sample of fresh muscle tissue is: freezing the fresh tissue into 5-8um slices on a glass slide. Further preferably, the method further comprises fixing the cells with analytical grade cold acetone for 5 minutes to prevent cell autolysis.

[0018] Preferably, in step S1, the method for preparing a glass slide sample of fresh muscle tissue is: cutting a cross section of the fresh muscle tissue, wiping off blood stains and printing the cross section on a glass slide, and then staining the cross section using the print on the glass slide.

[0019] Preferably, in step S2, the concentration of periodic acid is 10-15%. Further preferably, the concentration of periodic acid is 10%.

[0020] Preferably, in step S3, the ratio of the components of the striated muscle striae stain is: the mass ratio of the color-changing green 2R staining solution to methyl green is (3.6-4):1; the volume ratio of water to glacial acetic acid is (98-99):(1-2). Further preferably, in step S3, the formula of the striated muscle striae stain is: 3.6g of color-changing green 2R staining solution, 1g of methyl green, 99ml of water, and 1ml of glacial acetic acid.

[0021] Preferably, in step S3, the striated muscle striae stain is freshly prepared and used within 1 hour, and the binding force of the color-changing green 2R dye solution decreases over time.

[0022] Preferably, in step S3, the dyeing time is 5-8 minutes, and in step S5, the counter-dyeing time is 30-60 seconds. Further preferably, in step S3, the dyeing time is 5 minutes, and in step S5, the counter-dyeing time is 30 seconds.

[0023] Preferably, in step S5, the concentration of the aniline blue dye solution is 1-2%. Further preferably, in step S5, the concentration of the aniline blue dye solution is 1%.

[0024] The second aspect of the present invention provides a skeletal muscle striae stain, which comprises the following components: color-changing green 2R staining solution, methyl green, water, and glacial acetic acid; and the ratio of each component is: the mass ratio of color-changing green 2R staining solution to methyl green is (3.6-4):1; the volume ratio of water to glacial acetic acid is (98-99):(1-2).

[0025] Preferably, the striated muscle striae staining agent comprises the following formula: 3.6 g of color-changing green 2R staining solution, 1 g of methyl green, 99 ml of water, and 1 ml of glacial acetic acid.

[0026] Preferably, the striated muscle striae stain is freshly prepared and used within 1 hour, as the binding force of the color-changing green 2R dye solution decreases over time.

[0027] Compared with the prior art, the beneficial effects and significant progress of the technical solution of the present invention are: the striated muscle striated staining agent and staining method provided by the present invention, the color-changing green 2R dye solution combined with methyl green and the fresh striated muscle color band react specifically to show red, the striated muscle tissue aniline blue is stained into blue-green, and the remaining tissues such as the loose connective tissue between muscles are light green. The method of the present invention can show the coronal and sagittal planes of the muscle more clearly through the contrast between red and blue-green, and has the advantage of highlighting the striated effect more than the light and dark staining of the striated muscle staining in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments of the present invention are briefly introduced below.

[0029] Figure 1 It is a staining diagram of the Mallory phosphotungstic acid-hematoxylin-eosin staining method in the prior art;

[0030] Figure 2 It is a staining diagram of the Mallory phosphotungstic acid-hematoxylin-eosin staining method in the prior art;

[0031] Figure 3 is a staining diagram of the striated muscle striation staining method of Example 1;

[0032] Figure 4 It is the staining diagram of the striated muscle striation staining method of Example 2;

[0033] Figure 5 This is a staining diagram of the striated muscle striation staining method of comparative example 1. DETAILED DESCRIPTION

[0034] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0035] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0036] In order to more fully understand the present invention, the professional terms in the present invention are explained below.

[0037] Chromotropic Green 2R staining solution, also known as myelin staining solution (Chromotropic acid 2R method), is mainly used for staining myelin sheaths in pathological diagnosis in the prior art. This staining solution is used to show whether the myelin sheath is intact, degenerated, necrotic and repaired under pathological conditions, which is of great significance for the pathological diagnosis and research of nerve tissue. The myelin sheath is dark green after staining, while the demyelinated fibers are not stained.

[0038] Methyl green is a green micro-crystal or bright green powder with metallic luster. It is soluble in water and appears blue-green. It is slightly soluble in ethanol and insoluble in amyl alcohol. It is a basic dye that easily binds to DNA with a high degree of polymerization and appears green. It is also called double green SF. It is a green crystal with a golden luster or a light green powder. It is soluble in water and appears blue-green. It is slightly soluble in ethanol and insoluble in ether.

[0039] Glacial acetic acid, also known as acetic acid, acetic acid, chemical formula CH3COOH, is an organic monobasic acid, the source of the sour taste and pungent smell in vinegar. Pure anhydrous acetic acid (glacial acetic acid) is a colorless hygroscopic liquid with a freezing point of 16.7℃ (62℉), and it solidifies into colorless crystals. Although acetic acid is a weak acid based on its dissociation ability in aqueous solution, it is corrosive and its vapor is irritating to the eyes and nose.

[0040] Periodic acid, a white crystalline solid, usually exists in the form of a dihydrate. It is a strong oxidant that reacts with many organic and inorganic substances. Periodic acid dissolves in water to form an acidic solution, which is a strong acid. Colorless or white crystals, odorless, deliquescent; melting point 122°C; boiling point 140°C (decomposition); easily soluble in water, ethanol, slightly soluble in ether; density relative to air is 7.9; stability: stable; hazard mark 11 (oxidant). Uses in the prior art: Periodic acid is widely used in chemical experiments as an oxidant and catalyst. It can be used to synthesize other compounds, such as synthesizing ketone compounds or oxidizing alcohols to ketones. Periodic acid can also be used for selective oxidation reactions in organic synthesis. In organic chemistry, periodic acid can oxidize geminal diols and break carbon-carbon bonds to form two aldehydes, which are used to determine the structure of sugars.

[0041] Aniline blue dye is a commonly used dye with multiple uses and characteristics. Uses in the prior art: Aniline blue dye can be used to dye plant callose, using the principle that aniline blue specifically binds to plant callose and excites fluorescence at a specific wavelength. It is also used to dye nerve tissue, cells, connective tissue, and as an acid-base indicator. In pathology, aniline blue dye is used as one of the components of Masson's trichrome stain to distinguish collagen fibers from muscle fibers.

[0042] Re-staining is a technique used in the staining process. Its main purpose is to use a different dye to stain again based on the staining that has already been done once, in order to enhance the staining effect or distinguish different structures. Re-staining is an important part of the staining technique, which helps to improve the contrast and accuracy of the staining, making the results clearer and easier to analyze.

[0043] Example 1

[0044] In this example, the following method was used to stain the striations of striated muscle.

[0045] 1. Preparation before dyeing

[0046] 1.1. Preparation of slide samples of fresh muscle tissue

[0047] Fresh tissue can be frozen and cut into 5 μm slices on slides.

[0048] 1.2. Prepare the striated muscle stain

[0049] 3.6g of Chromochrome Green 2R staining solution, 1g of Methyl Green, 99ml of distilled water, 1ml of glacial acetic acid. The staining agent should be prepared fresh and used within 1 hour.

[0050] 2. Dyeing

[0051] 2.1. Wash the slides of fresh muscle tissue with 10% periodic acid oxidized water;

[0052] 2.2, staining with striated muscle stain;

[0053] 2.3. After 5 minutes of dyeing, rinse off the excess dye with running water. At this time, the dye color-changing green 2R combines with the horizontal stripes in the striated muscle to appear light red, and the overall background is light green;

[0054] 2.4. Add 1% aniline blue dye solution for counter-staining for 30 seconds so that the aniline blue dye solution combines with the color-changing green 2R dye combined with the striated muscle in the striated muscle and the color changes to purple-red, and the remaining striated muscle tissue is dyed blue;

[0055] 2.5. Finally, wash away the excess dye.

[0056] The dyeing results of this example are as follows Figure 3 As shown in the figure, the color-changing green 2R dye combined with methyl green reacts specifically with the fresh striated muscle band to show red, the striated muscle tissue is stained blue-green with aniline blue, and the other tissues such as the loose connective tissue between muscles are light green. The three colors are clearly distinguished in the figure.

[0057] Example 2

[0058] In this example, the following method was used to stain the striations of striated muscle.

[0059] 1. Preparation before dyeing

[0060] 1.1. Preparation of slide samples of fresh muscle tissue

[0061] Directly cut the fresh muscle tissue into sections, wipe off the blood stains and print them on a glass slide, then use the print on the slide for staining;

[0062] 1.2. Prepare the striated muscle stain

[0063] 3.6g of Chromochrome Green 2R staining solution, 1g of Methyl Green, 99ml of distilled water, 1ml of glacial acetic acid. The staining agent should be prepared fresh and used within 1 hour.

[0064] 2. Dyeing

[0065] 2.1. Wash the slides of fresh muscle tissue with 10% periodic acid oxidized water;

[0066] 2.2, staining with striated muscle stain;

[0067] 2.3. After 5 minutes of dyeing, rinse off the excess dye with running water. At this time, the dye color-changing green 2R combines with the horizontal stripes in the striated muscle to appear light red, and the overall background is light green;

[0068] 2.4. Add 1% aniline blue dye solution for counter-staining for 30 seconds so that the aniline blue dye solution combines with the color-changing green 2R dye combined with the striated muscle in the striated muscle and the color changes to purple-red, and the remaining striated muscle tissue is dyed blue;

[0069] 2.5. Finally, wash away the excess dye.

[0070] The dyeing results of this example are as follows Figure 4 As shown in the figure, the color-changing green 2R dye combined with methyl green reacts specifically with the fresh striated muscle band to show red, the striated muscle tissue is stained blue-green with aniline blue, and the other tissues such as the loose connective tissue between muscles are light green. The three colors are clearly distinguished in the figure.

[0071] Comparative Example 1

[0072] In this example, the following method was used to stain the striations of striated muscle.

[0073] 1. Preparation before dyeing

[0074] 1.1. Preparation of slide samples of fresh muscle tissue

[0075] Directly cut the fresh muscle tissue into sections, wipe off the blood stains and print them on a glass slide, then use the print on the slide for staining;

[0076] 1.2. Prepare the striated muscle stain

[0077] 3.6g of Chromochrome Green 2R staining solution, 1g of Methyl Green, 99ml of distilled water, 1ml of glacial acetic acid, let the stain sit for 2 hours before use.

[0078] 2. Dyeing

[0079] 2.1. Wash the slides of fresh muscle tissue with 10% periodic acid oxidized water;

[0080] 2.2, staining with striated muscle stain;

[0081] 2.3. Rinse off excess dye with running water after dyeing for 5 minutes;

[0082] 2.4. Add 1% aniline blue dye solution for counterstaining for 30 seconds;

[0083] 2.5. Finally, wash away the excess dye.

[0084] The dyeing results of this example are as follows Figure 5As shown, it can be seen that the staining effect is poor after the reagent is stored for 2 hours, and the ability to bind to the horizontal stripes is not strong.

[0085] The applicant declares that, in the description of the above specification:

[0086] The descriptions of the terms "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without causing any contradiction.

[0087] Finally, it should be noted that:

[0088] The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them.

[0089] 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 or all of the technical features therein, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection required by the present invention.

Claims

1. A method for staining striated muscle striations, characterized in that: The following steps are involved: S1. Prepare slide samples of fresh muscle tissue; S2, periodic acid oxidation washing; S3, staining with a striated muscle stain, wherein the striated muscle stain comprises the following components: color-changing green 2R staining solution, methyl green, water, and glacial acetic acid; S4, after dyeing, rinse off the excess dye with running water; S5, adding aniline blue dye solution for counter-dyeing; S6. Wash off excess dye.

2. A method for staining striated muscle striations as claimed in claim 1, characterized in that: In step S1, the method for preparing a glass slide sample of fresh muscle tissue is: freezing the fresh tissue and cutting it into 5-8 um slices on a glass slide.

3. A method for staining striated muscle striations as claimed in claim 1, characterized in that: In step S1, the method for preparing a glass slide sample of fresh muscle tissue is: cutting a cross section of the fresh muscle tissue, wiping off blood stains and printing the cross section on a glass slide, and then staining the cross section using the print on the glass slide.

4. A method for staining striated muscle striations as claimed in claim 1, characterized in that: In step S2, the concentration of periodic acid is 10-15%.

5. A method for staining striated muscle striations as claimed in claim 1, characterized in that: In step S3, the ratio of the components of the striated muscle striae stain is: the mass ratio of the color-changing green 2R staining solution to methyl green is (3.6-4):1; the volume ratio of water to glacial acetic acid is (98-99):(1-2).

6. A method for staining striated muscle striations as claimed in claim 1, characterized in that: In step S3, the striated muscle striae staining agent is freshly prepared and used within 1 hour. If used over time, the binding force of the color-changing green 2R dye solution decreases.

7. A method for staining striated muscle striations as claimed in claim 1, characterized in that: In step S3, the staining time is 5-8 minutes, and in step S5, the restaining time is 30-60 seconds.

8. A method for staining striated muscle striations as claimed in claim 1, characterized in that: In step S5, the concentration of the aniline blue dye solution is 1-2%.

9. A striated muscle striae staining agent, characterized in that: The striated muscle striae staining agent comprises the following components: color-changing green 2R staining solution, methyl green, water, and glacial acetic acid; and the proportions of the components are: the mass ratio of the color-changing green 2R staining solution to the methyl green is (3.6-4):1; the volume ratio of water to the glacial acetic acid is (98-99):(1-2).

10. A striated muscle staining agent as claimed in claim 9, characterized in that: The striated muscle striae staining agent is freshly prepared and used within 1 hour. If used overtime, the binding force of the color-changing green 2R dye solution decreases.

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