A reagent for staining pathological sections and its application

Abstract

The present application relates to the field of pathological detection technology, in particular to a reagent for staining pathological sections and its application. A reagent for staining pathological sections, including hematoxylin staining solution, the hematoxylin staining solution includes the following raw materials: 2-3g hematoxylin, 17-18g potassium aluminum sulfate, 0.15-0.25g sodium iodate, 240-260ml anhydrous ethanol, 700-800ml distilled water, 8-12ml glacial acetic acid, 0.1-0.2g hybrid hydrogel; the hybrid hydrogel is prepared by grafting amino clay on cyclodextrin. The present application adjusts the ratio of each raw material, makes full use of the oxidation and acid-base balance of the reagent itself, ensures the stability of the reagent, and adds a hybrid hydrogel to quickly adsorb iodine with a negative charge, reduces the influence of the reduction product of sodium iodate on staining, shortens the staining time, and improves the color development of the staining.

Description

Technical Field

[0001] The present application relates to the technical field of pathological detection, and in particular to a reagent for staining pathological sections and its application. Background Art

[0002] Pathological sections in a broad sense include diseased tissue sections and diseased cell samples. In a narrow sense, pathological sections are made by taking a certain size of diseased tissue and using pathological histological methods. The diseased tissue is usually embedded in a paraffin block and cut into thin slices using a slicer. When testing pathological sections, it is first stained with a stain, then the lesion is further examined under a microscope, and finally a pathological diagnosis is made.

[0003] The commonly used staining agent is hematoxylin-eosin staining agent. When using it, it is operated according to the instructions of the staining kit. During use, the applicant found that in actual use, the staining time of different batches is different, the effect is not enough (not blue enough), and the staining time is long, which takes 5-10 minutes. Insufficient staining color will lead to subsequent observation difficulties, and the required staining time is long. Therefore, improving the color development of staining is particularly important for improving work efficiency and detection accuracy. Summary of the invention

[0004] In order to improve the color development of staining while reducing the staining processing time, the present application provides a reagent for staining pathological sections and its application.

[0005] In a first aspect, the present application provides a reagent for staining pathological sections, which adopts the following technical solution.

[0006] A reagent for staining pathological sections, comprising a hematoxylin stain and an eosin stain, wherein the hematoxylin stain comprises the following raw materials:

[0007] Hematoxylin 2-3g, potassium aluminum sulfate 17-18g, sodium iodate 0.15-0.25g, anhydrous ethanol 240-260ml, distilled water 700-800ml, glacial acetic acid 8-12ml, hybrid hydrogel 0.1-0.2g;

[0008] The hybrid hydrogel is prepared by grafting amino clay onto cyclodextrin.

[0009] By adopting the above technical solution, the present application adjusts the ratio of each raw material, fully utilizes the oxidation and acid-base balance of the reagent itself, ensures the stability of the reagent, and improves the dyeing effect.

[0010] The dye sodium iodate of the present application plays a key role as an oxidant, which can oxidize hematoxylin to hematoxylin red, thereby giving the dyeing solution dyeing ability. However, after sodium iodate is reduced, the reduction product has a single iodide ion or iodine element, and these reduction products may participate in the complex reaction with hematoxylin and other components to form a variety of possible compounds, affecting the dyeing effect. In the present application, the hybrid hydrogel obtained by grafting amino clay in cyclodextrin is added to the hematoxylin dyeing solution. The hybrid hydrogel not only has a porous structure, but also introduces surface positive charge and cyclodextrin cavity on the surface of the hybrid hydrogel, so that the hybrid hydrogel has a very strong adsorption effect. During the dyeing process, the iodine with negative charge is quickly adsorbed, and the absorbed iodine is collected in the cavity, reducing the probability of iodine ions participating in the reaction of hematoxylin, thereby reducing the influence of the reduction product of sodium iodate on dyeing, shortening the dyeing time, and improving the color development of dyeing.

[0011] Furthermore, the preparation method of the hybrid hydrogel is:

[0012] 1) dissolving β-cyclodextrin to obtain a β-cyclodextrin solution;

[0013] Heat the β-cyclodextrin solution to 50-70°C, add the 1,4-butane sultone solution to the β-cyclodextrin solution while stirring, and react for 4-6 hours;

[0014] After the reaction is completed, hydrochloric acid is added to neutralize the alkaline substances in the reaction solution, and then the generated sulfobutyl-β-cyclodextrin is separated from the reaction solution by dialysis and ultrafiltration, and unreacted raw materials and by-products are removed;

[0015] The purified sulfobutyl-β-cyclodextrin solution is dried to obtain solid sulfonated cyclodextrin;

[0016] 2) Dissolve magnesium chloride in anhydrous ethanol, then add 3-aminopropyltriethoxysilane and stir to react for 24 hours, then centrifuge, wash and dry to obtain a white colloidal precipitate, i.e. amino clay;

[0017] 3) dissolving the obtained sulfonated cyclodextrin to obtain a sulfonated cyclodextrin solution, dissolving the amino clay to obtain an amino clay solution, mixing the sulfonated cyclodextrin solution and the amino clay solution for reaction for 2-3 hours to obtain a hybrid hydrogel.

[0018] By adopting the above technical scheme, β-cyclodextrin is sulfonated and sulfonate groups are introduced into β-cyclodextrin. The introduction of sulfonate groups makes the small end of cyclodextrin have a very high negative charge density. Amino clay is an organic clay with a sandwich structure. The upper and lower sides of the compound are modified with amino groups. The presence of amino groups makes its surface positively charged. The small end of cyclodextrin carries a negative charge with a high charge density. Amino clay forms a layer of exfoliated material with a positive charge on the surface in water. The two are cross-linked by electrostatic action to wrap water to form a hybrid hydrogel. The obtained hybrid hydrogel has the following characteristics: the surface has a porous structure, exhibits negative charge, and retains the cavity structure of cyclodextrin. It quickly adsorbs negatively charged iodine ions through the positive charge of the surface and the adsorption of cyclodextrin.

[0019] Furthermore, the weight ratio of the β-cyclodextrin to 1,4-butane sultone is (10-12):1.

[0020] Furthermore, in said 3), the weight ratio of sulfonated cyclodextrin to amino clay is 1:(5-7).

[0021] Furthermore, the weight percentage concentration of the sulfonated cyclodextrin solution in 3) is 2-3%, and the weight percentage concentration of the amino clay solution is 12-15%.

[0022] By adopting the above technical scheme, the amount of sulfonate groups grafted on cyclodextrin and the ratio of cyclodextrin to amino clay are within the ratio range specified in the present application, and the obtained hybrid hydrogel has a better adsorption effect on iodine ions, which can effectively control the adverse effects of iodine ions on the dyeing effect and improve the dyeing effect.

[0023] Furthermore, it also includes eosin staining solution, and the eosin staining solution includes the following raw materials:

[0024] Eosin 3-4g, anhydrous ethanol 900-1000ml, glacial acetic acid 2.5-3.5ml.

[0025] Furthermore, it also includes a brilliant green dye solution, which includes the following raw materials:

[0026] Brilliant green 2-3g, distilled water 15-25ml

[0027] Further, the preparation method of the hematoxylin stain solution is:

[0028] 1) Dissolving hematoxylin in anhydrous ethanol to obtain a hematoxylin solution;

[0029] 2) Dissolving potassium aluminum sulfate in distilled water to obtain a potassium aluminum sulfate solution;

[0030] 3) mixing the hematoxylin solution with the potassium aluminum sulfate solution to obtain a mixed solution;

[0031] 4) Add sodium iodate to the mixture. After the sodium iodate dissolves, add glacial acetic acid and mix to obtain the hematoxylin stain solution.

[0032] Furthermore, the preparation method of the eosin stain is:

[0033] a) dissolving eosin in anhydrous ethanol to obtain an eosin solution;

[0034] b) Add glacial acetic acid to the eosin solution and mix to obtain eosin staining solution.

[0035] Further, the preparation method of the brilliant green dye solution is:

[0036] Dissolve brilliant green in distilled water to obtain brilliant green dye solution.

[0037] In a second aspect, the present application provides an application of a reagent for staining pathological sections, using the following technical solution.

[0038] An application of a reagent for staining pathological sections, which is applied to staining tissue sections, comprises the following steps:

[0039] S1. Treat tissue sections with hematoxylin stain for 2-3 minutes;

[0040] S2. Treat with eosin solution for 10 seconds and the staining is complete.

[0041] Further, it is applied to cell staining, comprising the following steps:

[0042] S1. Treat cells with hematoxylin solution for 2-3 minutes;

[0043] S2. Treat with eosin solution for 3 minutes to complete the staining.

[0044] By adopting the above technical solution, the entire dyeing process can be completed in about 3 minutes, which greatly shortens the dyeing time and improves work efficiency.

[0045] In summary, this application has the following beneficial effects:

[0046] The present application adjusts the ratio of each raw material, makes full use of the oxidation and acid-base balance of the reagent itself, ensures the stability of the reagent, improves the dyeing effect, and adds a hybrid hydrogel obtained by grafting amino clay on cyclodextrin. The hybrid hydrogel not only has a porous structure, but also introduces surface positive charge and cyclodextrin cavity on the surface of the hybrid hydrogel, so that the hybrid hydrogel has a strong adsorption effect. During the dyeing process, it quickly adsorbs negatively charged iodine and takes the absorbed iodine into the cavity, reducing the probability of iodine ions participating in the reaction of hematoxylin, thereby reducing the influence of the reduction product of sodium iodate on dyeing, shortening the dyeing time, and improving the color rendering of the dyeing. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the dyeing effect diagram of Application Example 1;

[0048] Figure 2 This is the dyeing effect diagram of Application Example 2;

[0049] Figure 3 This is the dyeing effect diagram of Application Example 3;

[0050] Figure 4 This is the dyeing effect diagram of Application Example 4;

[0051] Figure 5 This is a dyeing effect diagram of Application Example 5;

[0052] Figure 6 This is a dyeing effect diagram of Application Example 6;

[0053] Figure 7 This is the dyeing effect diagram of Application Example 7;

[0054] Figure 8 This is the dyeing effect diagram of Application Example 8;

[0055] Fig. 9 This is a dyeing effect diagram of Application Example 9;

[0056] Fig.10 This is a dyeing effect diagram of comparative application example 1;

[0057] Fig.11 This is a diagram of the dyeing effect of comparative application example 2. DETAILED DESCRIPTION

[0058] The present application is further described in detail below with reference to the embodiments.

[0059] Raw materials and intermediate preparation examples

[0060] raw material

[0061] The raw materials in the examples of this application can be obtained from the market:

[0062] Hematoxylin, CAS No. 517-28-2, analytical grade, powder;

[0063] Potassium aluminum sulfate, analytical grade;

[0064] Sodium iodate, analytical grade;

[0065] Anhydrous ethanol, analytical grade;

[0066] Glacial acetic acid, analytical grade

[0067] Eosin, alcohol soluble, powder reagent;

[0068] Brilliant green, CAS No. 633-03-4, analytical grade;

[0069] β-cyclodextrin, CAS No. 7585-39-9, molecular weight 1134.98, powder, effective content 99%;

[0070] 1,4-Butane sultone, analytical grade;

[0071] Hydrochloric acid, analytical grade;

[0072] Magnesium chloride, analytical grade;

[0073] 3-Aminopropyltriethoxysilane, analytical grade;

[0074] The mass concentration of sodium hydroxide solution is 4%.

[0075] Preparation Example

[0076] Preparation Example 1

[0077] A hybrid hydrogel, the preparation method of which is:

[0078] 1) Dissolve 100 g of β-cyclodextrin in 1 L of sodium hydroxide solution to obtain a β-cyclodextrin solution;

[0079] Heat the β-cyclodextrin solution to 60°C, add 10 g of 1,4-butane sultone into the β-cyclodextrin solution while stirring, and react for 5 hours;

[0080] After the reaction is completed, hydrochloric acid is added to neutralize the alkaline substances in the reaction solution until the pH value of the reaction solution reaches 7, and then dialysis and ultrafiltration are used to separate and purify the generated sulfobutyl-β-cyclodextrin from the reaction solution, and unreacted raw materials and by-products are removed;

[0081] The purified sulfobutyl-β-cyclodextrin was dried at 80° C. for 3 h to obtain sulfonated cyclodextrin in solid form;

[0082] 2) Dissolve 17g of magnesium chloride in 500ml of anhydrous ethanol, then add 25g of 3-aminopropyltriethoxysilane and stir to react for 24h, then centrifuge, wash and dry to obtain a white colloidal precipitate, i.e. amino clay;

[0083] 3) Dissolve 10 g of the obtained sulfonated cyclodextrin in water to prepare a sulfonated cyclodextrin solution with a weight percentage concentration of 2.5%, dissolve 60 g of amino clay in water to obtain an amino clay solution with a weight percentage concentration of 13%, mix the sulfonated cyclodextrin solution and the amino clay solution for 3 hours to obtain a hybrid hydrogel.

[0084] Preparation Example 2

[0085] Different from Preparation Example 1, in step 3) of Preparation Example 2, 10 g of the obtained sulfonated cyclodextrin was dissolved in water to prepare a sulfonated cyclodextrin solution with a weight percentage concentration of 2.5%, 50 g of amino clay was dissolved in water to obtain an amino clay solution with a weight percentage concentration of 13%, and the sulfonated cyclodextrin solution and the amino clay solution were mixed and reacted for 3 hours to obtain a hybrid hydrogel.

[0086] Preparation Example 3

[0087] Different from Preparation Example 1, in step 3) of Preparation Example 2, 10 g of the obtained sulfonated cyclodextrin was dissolved in water to prepare a sulfonated cyclodextrin solution with a weight percentage concentration of 2.5%, 70 g of amino clay was dissolved in water to obtain an amino clay solution with a weight percentage concentration of 13%, and the sulfonated cyclodextrin solution and the amino clay solution were mixed and reacted for 3 hours to obtain a hybrid hydrogel. Example

[0088] Examples 1-4

[0089] A reagent for staining pathological sections, the preparation method of which is as follows:

[0090] Preparation of hematoxylin stain:

[0091] 1) Dissolve hematoxylin in anhydrous ethanol according to the raw material ratio in Table 1 to obtain a hematoxylin solution;

[0092] 2) Dissolving potassium aluminum sulfate in distilled water to obtain a potassium aluminum sulfate solution;

[0093] 3) mixing the hematoxylin solution with the potassium aluminum sulfate solution to obtain a mixed solution;

[0094] 4) Add sodium iodate to the mixture. After the sodium iodate is dissolved, add glacial acetic acid and mix to obtain a hematoxylin stain solution;

[0095] The preparation method of the brilliant green dye solution is:

[0096] Dissolve brilliant green in distilled water to obtain brilliant green dye solution.

[0097] Table 1 Raw material ratio table of Examples 1-4

[0098]

[0099] Among them, the hybrid hydrogel comes from Preparation Example 1.

[0100] Embodiment 5-6

[0101] Different from Example 2, the hybrid hydrogels in Examples 5-7 are from Preparation Examples 2-3, respectively.

[0102] Example 7

[0103] Different from Example 2, in Example 7, an equal amount of β-cyclodextrin was used to replace the hybrid hydrogel.

[0104] Example 8

[0105] A reagent for staining pathological sections, the preparation method of which is as follows:

[0106] Preparation of lignin dye solution:

[0107] Same as Example 2;

[0108] The preparation method of the eosin stain is:

[0109] a) Dissolve 3.5 g of eosin in 950 ml of anhydrous ethanol to obtain an eosin solution;

[0110] b) Add 3 ml of glacial acetic acid to the eosin solution and mix to obtain eosin staining solution.

[0111] Example 9

[0112] Different from Example 8, the hematoxylin staining solution in Example 9 is the same as that in Example 4.

[0113] Comparative Example

[0114] Comparative Example 1

[0115] Different from Example 1, the hematoxylin staining solution in Comparative Example 1 is the hematoxylin staining solution in the HE staining solution with the product number M020 of Shanghai Gefan Biological.

[0116] Application Examples

[0117] Application Example 1

[0118] Staining of cervical cells includes the following steps:

[0119] S1. Wash with water for 10 seconds;

[0120] S2. Treat the cervical cell sample with the hematoxylin stain in Example 1 for 2.5 min;

[0121] S3. Wash with water for 30 seconds;

[0122] S4. Treat with hydrochloric acid ethanol differentiation solution for 1 second;

[0123] S5. Wash with water for 1 min;

[0124] S6. Wash with 95% ethanol for 30 seconds;

[0125] S7. Orange-G solution staining for 5 seconds;

[0126] S8.95% ethanol treatment twice, the first time for 20 seconds, the second time for 30 seconds;

[0127] S9. Treat with the brilliant green dye solution in Example 1 for 3 min;

[0128] S10. Treat with 95% ethanol twice, 30 seconds each time; then treat with anhydrous ethanol twice, 1 minute for the first time and 2 minutes for the second time.

[0129] Application Example 2-7

[0130] Different from Application Example 1, the dyes in Application Examples 2-7 are respectively from Examples 2-7.

[0131] Application Example 8

[0132] The staining of uterine leiomyoma sections includes the following steps:

[0133] S1. Treat the uterine leiomyoma slices with xylene for 5 min, 5 min, 5 min, and 10 min in sequence;

[0134] S2. Treat with anhydrous ethanol twice, 1 min each time; then treat with 95% ethanol for 50 s; and then treat with 75% ethanol for 50 s;

[0135] S3. Wash with water for 1 min;

[0136] S4. Treat the cervical cell sample with the hematoxylin stain in Example 8 for 2.5 min;

[0137] S5. Wash with water for 1 min;

[0138] S6. Treat with hydrochloric acid ethanol differentiation solution for 1 second;

[0139] S7. Wash with water for 2 minutes;

[0140] S8. The eosin staining solution in Example 8 was treated for 10 seconds;

[0141] S9. Wash with water for 5 seconds;

[0142] S10. Treat with 75% ethanol twice, the first time for 15 s and the second time for 20 s; then treat with 95% ethanol twice, the first time for 30 s and the second time for 30 s; finally treat with anhydrous ethanol for 1 min.

[0143] Application Example 9

[0144] Different from Application Example 8, the dye in Application Example 9 comes from Example 9.

[0145] Comparative Application Examples

[0146] Comparative application example 1

[0147] The difference from Example 1 is that the dye in Comparative Example 1 comes from Comparative Example 1, and the dyeing time is 10 minutes.

[0148] Comparative Application Example 2

[0149] The HE staining solution with the product number M020 from Shanghai Gefan Biotechnology was used to stain the uterine leiomyoma sections according to the operating instructions.

[0150] Performance Testing

[0151] The corresponding examples and comparative examples of sample dyeing are recorded in the attached Figure 1-11 .

[0152] Combined with Figure 1-7 With attached Fig.10 As well as application examples 1-7 and comparative application example 1, it can be seen that compared with the attached Fig.11 , attached Figure 1-7 The cervical cell staining smear has bright colors, and the colors of the cell nucleus and cytoplasm are distinct, which shows that the staining effect of cervical cells in Application Example 1-7 is better.

[0153] Combined with Figure 1-3 As can be seen from the application examples 1-3, Figure 1-3 The cervical cell staining smears are brightly colored, with distinct nuclei and cytoplasm. Figure 2 This shows that the ratio of the dye used in Application Example 2 is relatively better.

[0154] Combined with Figure 2 , Attachment Figure 4 With attached Figure 7 As for Application Examples 2, 4 and 7, it can be seen that the cervical cell staining smears are bright in color, and the nucleus and cytoplasm are clearly colored. Figure 2 > Attached Figure 7 > Attached Figure 4 This shows that the dyeing effect of the dye used in Application Example 2 is better than that of Application Example 4, and the dyeing effect of the dye used in Application Example 4 is better than that of Application Example 7. This may be because the addition of hybrid hydrogel or β-cyclodextrin can absorb the reduction product of sodium iodate, thereby reducing the influence of the reduction product of sodium iodate on dyeing, shortening the dyeing time, and improving the color development of the dyeing. Among them, the hybrid hydrogel has a better adsorption effect on iodide ions than β-cyclodextrin.

[0155] Attached Figure 8-9 It was verified that the staining agent of the present application can be applied to uterine leiomyoma sections with excellent staining effect.

[0156] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A reagent for staining pathological sections, comprising a hematoxylin stain solution, characterized in that: The hematoxylin stain solution comprises the following raw materials: Hematoxylin 2-3g, potassium aluminum sulfate 17-18g, sodium iodate 0.15-0.25g, anhydrous ethanol 240-260ml, distilled water 700-800ml, glacial acetic acid 8-12ml, hybrid hydrogel 0.1-0.2g; The hybrid hydrogel is prepared by grafting amino clay onto cyclodextrin.

2. A reagent for staining pathological sections according to claim 1, characterized in that: The preparation method of the hybrid hydrogel is: 1) dissolving β-cyclodextrin to obtain a β-cyclodextrin solution; Heat the β-cyclodextrin solution to 50-70°C, add 1,4-butane sultone to the β-cyclodextrin solution while stirring, and react for 4-6 hours; After the reaction is completed, hydrochloric acid is added to neutralize the alkaline substances in the reaction solution, and then the generated sulfobutyl-β-cyclodextrin is separated from the reaction solution by dialysis and ultrafiltration, and unreacted raw materials and by-products are removed; The purified sulfobutyl-β-cyclodextrin is dried to obtain a solid sulfonated cyclodextrin; 2) Dissolve magnesium chloride in anhydrous ethanol, then add 3-aminopropyltriethoxysilane and stir to react for 24 hours, then centrifuge, wash and dry to obtain a white colloidal precipitate, i.e. amino clay; 3) dissolving the obtained sulfonated cyclodextrin to obtain a sulfonated cyclodextrin solution, dissolving the amino clay to obtain an amino clay solution, mixing the sulfonated cyclodextrin solution and the amino clay solution for reaction for 2-3 hours to obtain a hybrid hydrogel.

3. A reagent for staining pathological sections according to claim 2, characterized in that: The weight ratio of the β-cyclodextrin to 1,4-butane sultone is (10-12):

1.

4. A reagent for staining pathological sections according to claim 3, characterized in that: The weight ratio of sulfonated cyclodextrin to amino clay in the above 3) is 1:(5-7).

5. A reagent for staining pathological sections according to claim 4, characterized in that: The weight percentage concentration of the sulfonated cyclodextrin solution in the above 3) is 2-3%, and the weight percentage concentration of the amino clay solution is 12-15%.

6. A reagent for staining pathological sections according to claim 1, characterized in that: Also included is eosin staining solution, which includes the following raw materials: Eosin 3-4g, anhydrous ethanol 900-1000ml, glacial acetic acid 2.5-3.5ml.

7. A reagent for staining pathological sections according to claim 1, characterized in that: Also included is a brilliant green dye solution, which comprises the following raw materials: Brilliant green 2-3g, distilled water 15-25ml.

8. A use of the reagent for staining pathological sections as claimed in claim 6, characterized in that: It is applied to tissue section staining, including the following steps: S1. Treat tissue sections with hematoxylin stain for 2-3 minutes; S2. Treat with eosin solution for 10 seconds and the staining is complete.

9. A use of the reagent for staining pathological sections as claimed in claim 7, characterized in that: It is applied to cell staining and includes the following steps: S1. Treat cells with hematoxylin solution for 2-3 minutes; S2. Treat with Brilliant Green for 3 minutes and the staining is complete.

Images