Melanin decoloration reaction liquid and melanin decoloration method

Abstract

The invention belongs to the technical field of biological dyeing, and particularly relates to melanin decoloration reaction liquid and a melanin decoloration method. The melanin decolorization reaction liquid is an aqueous solution containing the following components in percentage by mass: 0.6-1.5% of hydrogen peroxide and 0.5-0.8% of strong base. The melanin decolorization method comprises the following steps: immersing a tissue slice into the melanin decolorization reaction solution, and incubating at 25-45 DEG C for 20-40 minutes. The method has the advantages that the decoloration speed is high, the temperature condition is mild, the tissue structure form after decoloration is complete, good antigenicity is kept, and DNA is not affected, and fluorescence in-situ hybridization can be performed after decoloration. Therefore, the method has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological staining, and particularly relates to a melanin decolorization reaction solution and a melanin decolorization method. Background Art

[0002] In the preparation of pathological tissue sections, pigment deposits are often encountered. These pigment deposits cause poor staining in a series of subsequent operations such as conventional staining, special staining, immunohistochemistry, and in-situ hybridization, which will directly affect the quality of staining and cause certain difficulties in diagnosis. Melanin is the most common pigment, which is mostly found in melanocytes of the eyes and skin, and is also prone to produce melanin in certain diseases of the body. Melanin, literally a black pigment, usually does not appear black under a microscope, but yellow-brown or brownish-yellow, and its particle size varies. This color is similar to the yellow-brown DAB chromogenic agent commonly used in immunohistochemical staining. A large amount of melanin deposition in tissues rich in melanoma will cover the normal tissue and cell morphology. Therefore, depigmentation treatment has become an important measure.

[0003] Melanin is an indole polymer derived from a series of chemical reactions of multiple tyrosines. Its basic structure is some indole rings obtained by covalent cross-linking. The indole regions of the indole polymer overlap with each other under the action of van der Waals forces, making the properties of melanin stable. It is insoluble in water and organic solvents and can be decolorized by strong oxidants. The hydroxyl group on the indole ring can undergo an oxidation-reduction reaction with the oxidant to generate a carboxyl group.

[0004] Potassium permanganate has strong oxidizing properties and can decolorize melanin. Therefore, the potassium permanganate / oxalic acid method for depigmentation is the most widely used classic oxidant depigmentation method at present. However, after decolorization treatment by the potassium permanganate / oxalic acid method, uneven decolorization can be seen in immunohistochemical staining, and partial tissue morphology is dissolved, the tissue antigenicity is reduced, and it has a greater impact on DNA, so fluorescence in-situ hybridization detection cannot be performed after decolorization. And after the tissue is treated with potassium permanganate solution, the cytoplasmic PH moves to the alkaline side, the cytoplasm is lightly stained, and the contrast between nuclear and cytoplasmic staining is not obvious.

[0005] Hydrogen peroxide is also an oxidant and can also be used for melanin decolorization. And it has basically no effect on the antigenicity of tissues at room temperature. However, the hydrogen peroxide decolorization method requires high concentration and high temperature conditions for decolorization and the decolorization time is long. Increasing the temperature of tissue section decolorization cannot significantly reduce the decolorization duration, and with the increase of temperature, the possibility of tissue section peeling is increased, which limits the application of this method. Therefore, there is an urgent need in the art to improve the existing melanin decolorization methods, so as to achieve rapid and mild-condition melanin decolorization on the premise of having less impact on tissue antigenicity. Summary of the Invention

[0006] In view of the problems of the prior art, the present invention provides a melanin decolorization reaction solution and a melanin decolorization method.

[0007] A melanin decolorization reaction solution is an aqueous solution containing the following components by mass fraction:

[0008] Hydrogen peroxide 0.6 - 1.5%,

[0009] Strong base 0.5 - 0.8%.

[0010] Preferably, it is an aqueous solution containing the following components by mass fraction:

[0011] Hydrogen peroxide 0.6%,

[0012] Strong base 0.8%.

[0013] Preferably, the strong base is selected from at least one of potassium hydroxide and sodium hydroxide.

[0014] Preferably, the melanin decolorization reaction solution is obtained by mixing a hydrogen peroxide solution and a strong base solution and incubating at 25 - 45 °C for 20 - 40 minutes.

[0015] Preferably, the melanin decolorization reaction solution uses a hydrothermal solution of 0.6% hydrogen peroxide and 0.8% strong base by mass fraction.

[0016] The present invention also provides a melanin decolorization method, including the following steps: immersing a tissue section in the above melanin decolorization reaction solution and incubating at 25 - 45 °C for 20 - 40 minutes.

[0017] Preferably, the incubation conditions are: incubating at 25 - 45 °C for 20 - 40 minutes.

[0018] The present invention also provides a staining method, including the following steps:

[0019] Step 1, performing melanin decolorization on the tissue section according to the above melanin decolorization method;

[0020] Step 2, staining the tissue section after melanin decolorization.

[0021] Preferably, the staining is HE staining after decolorization or immunohistochemical staining after decolorization.

[0022] The present invention selects strong bases such as potassium hydroxide as catalysts to accelerate the decomposition of hydrogen peroxide, improve the oxidation efficiency, thereby increasing the decolorization speed of the hydrogen peroxide method for melanin decolorization in the prior art, and thus being able to reduce the processing time without using a relatively high temperature. On the other hand, hydrogen peroxide has acidity (for example, 3% H 2 O 2(with a pH of 3.5), which shifts the pH of the tissue environment to the acidic side. Both the cytoplasm and the nucleus exhibit basic ionization, carry positive charges, and are stained by acidic dyes. As a result, the staining of the cytoplasm and the nucleus cannot be distinguished, and acids are prone to corrode, causing irreversible effects on DNA. In the present invention, an alkali is added to the hydrogen peroxide solution to prepare a decolorization reaction solution, which can change and balance the pH of the solution. After decolorization, subsequent staining is clearer, and DNA can be well preserved. The specimens after decolorization can still be used for fluorescence in situ hybridization and gene detection, and have advantages in application fields such as the diagnosis of melanoma.

[0023] In addition, in the preferred embodiment, the present invention optimizes the preparation method of the decolorization reaction solution. On the one hand, by pre-incubating and preheating after mixing, H 2 O 2 is in a decomposed state to reduce the decolorization time; on the other hand, the incubation time is controlled to take into account that H 2 O 2 does not completely decompose and lose the decolorization effect.

[0024] In summary, the present invention has good application prospects in the staining of biological tissue sections.

[0025] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0026] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0027] Figure 1 is the experimental result of HE staining without decolorization in Experimental Example 1;

[0028] Figure 2 is the experimental result of staining after decolorization by the potassium permanganate-oxalic acid method in Experimental Example 1;

[0029] Figure 3 is the experimental result of staining after decolorization according to the method of the present invention in Experimental Example 1; Figure 4 is the experimental result of the screening of the concentration ratio of 3% hydrogen peroxide to 1% potassium hydroxide in Experimental Example 2. Specific Embodiments

[0030] In the following examples and experimental examples, the reagents and raw materials not specifically described are all commercially available products.

[0031] Example 1 Melanin Decolorization Reaction Solution

[0032] The melanin decolorization reaction solution of this example is an aqueous solution containing the following components by mass fraction:

[0033] Hydrogen peroxide 0.6%,

[0034] Potassium hydroxide 0.8%.

[0035] The melanin decolorization reaction solution is prepared by the following method according to the principle of preparing and using it immediately:

[0036] Mix the freshly prepared hydrogen peroxide solution with a mass fraction of 3% and the potassium hydroxide solution with a mass fraction of 1% according to a volume ratio of 1:4, and incubate in an incubator at 37°C for 30 minutes before use.

[0037] Melanin decolorization method in Example 2

[0038] This example uses the melanin decolorization reaction solution prepared in Example 1 for melanin decolorization, which specifically includes the following steps:

[0039] (1) Fix the tissue with neutral formaldehyde solution, dehydrate, clear, and embed in paraffin routinely. The sections are dewaxed with xylene I for 15 minutes and xylene II for 15 minutes;

[0040] (2) Wash off the xylene with absolute ethanol for 1 minute, 95% ethanol for 1 minute, 90% ethanol for 1 minute, 85% ethanol for 1 minute, and wash with tap water for 2 minutes;

[0041] (3) Prepare the melanin decolorization reaction solution according to the method described in Example 1, immerse the tissue sections in the melanin decolorization reaction solution, and incubate at 37°C for 20 - 40 minutes. In this example, it is preferably incubated for 30 minutes.

[0042] (4) Observe under the microscope that the melanin has completely faded, and wash with water for 5 - 10 minutes.

[0043] HE staining method after melanin decolorization in Example 3

[0044] The staining method provided in this example includes the following steps:

[0045] (1) Perform melanin decolorization according to the method of Example 2;

[0046] (2) Stain with Harris hematoxylin for 5 minutes and wash with tap water for 1 minute;

[0047] (3) Differentiate with 1% hydrochloric acid ethanol for 30 seconds, wash with tap water for 5 minutes, and turn blue;

[0048] (4) Stain with 1% eosin for 1 minute and wash with tap water;

[0049] (5) Dehydrate, clear, and mount routinely.

[0050] Example 4 Immunohistochemical Staining Method after Melanin Decolorization

[0051] The staining method provided in this example includes the following steps:

[0052] (1) Perform melanin decolorization according to the method of Example 2;

[0053] (2) Select the corresponding method for repair according to the antibody (in this example, for SOX10 and HMB45, use EDTA repair solution and heat in a water bath at above 95 °C for 25 minutes);

[0054] (3) Drop the primary antibodies SOX10 and HMB45 and incubate in an incubator at 37 °C for 1 hour;

[0055] (4) Wash three times with PBS, 5 minutes each time;

[0056] (5) Drop the alkaline phosphatase-labeled secondary antibody and incubate in an incubator at 37 °C for 30 minutes;

[0057] (6) Wash three times with PBS, 5 minutes each time;)

[0058] (7) Select the AP chromogenic agent according to the secondary antibody for chromogenic reaction for 5 minutes, and wash with tap water;

[0059] (8) Counterstain with hematoxylin, dehydrate routinely, clear and mount.

[0060] The technical solution of the present invention will be further described below through experiments.

[0061] Experimental Example 1 Comparison of Staining Effects of Different Melanin Decolorization Methods

[0062] I. Experimental Method

[0063] 1. The operation steps of HE staining without decolorization for the control group in this experimental example are as follows:

[0064] (1) Fix the tissue with neutral formaldehyde solution, dehydrate, clear and embed in paraffin routinely; dewax the sections with xylene I for 15 minutes and xylene II for 15 minutes;

[0065] (2) Wash off xylene with absolute ethanol for 1 minute, 95% ethanol for 1 minute, 90% ethanol for 1 minute, 85% ethanol for 1 minute, and wash with tap water for 2 minutes;

[0066] (3) Stain with Harris hematoxylin for 5 minutes and wash with tap water for 1 minute;

[0067] (4) Differentiate with 1% hydrochloric acid ethanol for 30 seconds, wash with tap water for 5 minutes and turn blue;

[0068] (5) Stain with 1% eosin for 1 minute and wash with tap water;

[0069] (6) Conventional dehydration, clearing and mounting.

[0070] 2. The operation steps of decolorization by potassium permanganate - oxalic acid method in the control group of this experimental example are as follows:

[0071] Decolorization by potassium permanganate - oxalic acid method: The sections are dewaxed to water, immersed in 0.25% potassium permanganate solution for 1 - 3 hours, washed with water, then treated with 1% oxalic acid for 1 minute, and subjected to conventional staining. After decolorization, wash with water for 5 - 10 minutes, stain according to need, and perform conventional dehydration, clearing and mounting. During the oxidative bleaching process, the sections should be taken out for microscopic examination at any time to ensure that the melanin in the cells is completely disappeared.

[0072] Steps of staining with HE after potassium permanganate fading:

[0073] (1) Fix the tissue with neutral formaldehyde solution, perform conventional dehydration, clearing, and paraffin embedding. The sections are dewaxed with xylene Ⅰ for 15 minutes and xylene Ⅱ for 15 minutes; wash off the xylene with absolute ethanol for 1 minute, 95% ethanol for 1 minute, 90% ethanol for 1 minute, 85% ethanol for 1 minute, and wash with tap water for 2 minutes;

[0074] (2) Immerse in 0.25% potassium permanganate solution for 1 - 3 hours, and wash with tap water for 5 minutes;

[0075] (3) Treat with 1% oxalic acid for 1 minute, observe under the microscope until the melanin has completely faded, and wash with water for 5 - 10 minutes

[0076] (4) Stain with Harris hematoxylin for 5 minutes, and wash with tap water for 1 minute;

[0077] (5) Differentiate with 1% hydrochloric acid ethanol for 30 seconds, wash with tap water for 5 minutes to turn blue;

[0078] (6) Stain with 1% eosin for 1 minute, and wash with tap water;

[0079] (7) Conventional dehydration, clearing and mounting.

[0080] Steps of immunohistochemical staining after potassium permanganate fading:

[0081] (1) Fix the tissue with neutral formaldehyde solution, perform conventional dehydration, clearing, and paraffin embedding. The sections are dewaxed with xylene Ⅰ for 15 minutes and xylene Ⅱ for 15 minutes; wash off the xylene with absolute ethanol for 1 minute, 95% ethanol for 1 minute, 90% ethanol for 1 minute, 85% ethanol for 1 minute, and wash with tap water for 2 minutes;

[0082] (2) Immerse in 0.25% potassium permanganate solution for 1 - 3 hours, and wash with tap water for 5 minutes;

[0083] (3) Treat with 1% oxalic acid for 1 minute, observe under the microscope that the melanin has completely faded, and wash with water for 5 - 10 minutes;

[0084] (4) Select the corresponding method for antigen retrieval according to the antibody (in our laboratory, for SOX10 and HMB45, the EDTA retrieval solution is used for water bath heating at over 95 °C for 25 minutes);

[0085] (3) Dropwise add the primary antibodies SOX10 and HMB45 and incubate in an incubator at 37 °C for 1 hour;

[0086] (4) Wash three times with PBS, 5 minutes each time;

[0087] (5) Dropwise add the alkaline phosphatase - labeled secondary antibody and incubate in an incubator at 37 °C for 30 minutes;

[0088] (6) Wash three times with PBS, 5 minutes each time;

[0089] (7) Select the AP chromogenic agent for chromogenic reaction for 5 minutes according to the secondary antibody, and wash with tap water;

[0090] (8) Counterstain with hematoxylin, dehydrate routinely, clear and mount.

[0091] 3. The experimental groups in this experimental example were respectively subjected to HE staining according to the method of Example 3, immunohistochemical SOX10 staining and immunohistochemical HMB45 staining according to the method of Example 4.

[0092] II. Experimental Results

[0093] The experimental results of HE staining without decolorization are as Figure 1 shown. It can be seen from the figure that the presence of melanin seriously affects the staining effect.

[0094] The staining results after decolorization by the potassium permanganate - oxalic acid method are as Figure 2 shown. It can be seen from the figure that the tissue structure has dissolved and disappeared, and the antigenicity of the antibody has also weakened.

[0095] The results of HE staining, immunohistochemical SOX10 staining and immunohistochemical HMB45 staining carried out according to the methods of Example 3 and Example 4 are as Figure 3 shown. It can be seen from the figure that the melanin has completely disappeared and the tissue structure is intact, and the tissue maintains good antigenicity, indicating that the method of the present invention does not affect DNA and fluorescence in situ hybridization can be carried out after decolorization.

[0096] Experimental Example 2 Screening Test of the Concentration Ratio of 3% Hydrogen Peroxide to 1% Potassium Hydroxide

[0097] I. Experimental Method

[0098] Select a specimen containing melanin, and make 9 consecutive sections. Dewax the sections to water; take 1 out of the 9 sections without melanin decolorization treatment; the remaining sections are treated as follows: 1 section is treated with 3% hydrogen peroxide; 1 section is treated with a decolorization reaction solution prepared by mixing 75 ml of 3% hydrogen peroxide and 25 ml of 1% potassium hydroxide in a ratio of 3:1; 1 section is treated with a decolorization reaction solution prepared by mixing 50 ml of 3% hydrogen peroxide and 50 ml of 1% potassium hydroxide in a ratio of 2:2; 1 section is treated with a decolorization reaction solution prepared by mixing 25 ml of 3% hydrogen peroxide and 75 ml of 1% potassium hydroxide in a ratio of 1:3; 1 section is treated with a decolorization reaction solution prepared by mixing 20 ml of 3% hydrogen peroxide and 80 ml of 1% potassium hydroxide in a ratio of 1:4; 1 section is treated with a decolorization reaction solution prepared by mixing 10 ml of 3% hydrogen peroxide and 90 ml of 1% potassium hydroxide in a ratio of 1:9; 1 section is treated with 1% potassium hydroxide:. All reaction solutions are pre-incubated in an incubator at 37°C for 30 minutes before use. The sections are decolorized in the reaction solution for 30 minutes, and the results are as follows.

[0099] II. Experimental Results

[0100] Experimental results: The results are as Figure 4 , it can be seen that 3% hydrogen peroxide cannot remove melanin in a short time; when the reaction solution prepared by mixing 75 ml of 3% hydrogen peroxide and 25 ml of 1% potassium hydroxide can remove part of the melanin within 30 minutes; the reaction solution prepared by mixing 50 ml of 3% hydrogen peroxide and 50 ml of 1% potassium hydroxide can almost completely remove melanin; the reaction solution prepared by mixing 25 ml of 3% hydrogen peroxide and 75 ml of 1% potassium hydroxide can remove melanin well; the reaction solution prepared by mixing 20 ml of 3% hydrogen peroxide and 80 ml of 1% potassium hydroxide can achieve a good effect of removing melanin; the effect of removing melanin by the reaction solution prepared by mixing 10 ml of 3% hydrogen peroxide and 90 ml of 1% potassium hydroxide is weakened; 1% potassium hydroxide also cannot remove melanin within 30 minutes.

[0101] According to the above experimental results, when the concentration of hydrogen peroxide in the decolorization reaction solution is 0.6 - 1.5% and the concentration of potassium hydroxide is 0.5 - 0.8%, the decolorization reaction can be preferably completed within 20 - 40 minutes in an incubator at 37°C.

[0102] From the above examples and experimental examples, it can be seen that the present invention provides a new melanin decolorization reaction solution and a melanin decolorization method, which have the advantages of fast decolorization speed, mild temperature conditions, and the integrity of the tissue structure and morphology after decolorization, maintaining good antigenicity, and not affecting DNA. Fluorescence in situ hybridization can be carried out after decolorization. Therefore, the present invention has good application prospects.

Claims

1. A melanin decolorization reaction solution, characterized in that, it is an aqueous solution containing components with the following mass fractions: hydrogen peroxide 0.6 - 1.5%, strong base 0.5 - 0.8%.

2. The melanin decolorization reaction solution according to claim 1, characterized in that: it is an aqueous solution containing components with the following mass fractions: hydrogen peroxide 0.6%, strong base 0.8%.

3. The melanin decolorization reaction solution according to claim 1, characterized in that: the strong base is selected from at least one of potassium hydroxide and sodium hydroxide.

4. The melanin decolorization reaction solution according to claim 1, characterized in that: the melanin decolorization reaction solution is obtained by mixing a hydrogen peroxide solution and a strong base solution and incubating at 25 - 45°C for 20 - 40 minutes.

5. The melanin decolorization reaction solution according to claim 1 or 4, characterized in that: the melanin decolorization reaction solution uses a hydrothermal solution of 0.6% hydrogen peroxide and 0.8% strong base by mass fraction.

6. A melanin decolorization method, characterized in that, comprises the following steps: immersing a tissue section in the melanin decolorization reaction solution according to any one of claims 1 - 4 and incubating at 25 - 45°C for 20 - 40 minutes.

7. The melanin decolorization method according to claim 6, characterized in that, the incubation conditions are: incubating at 25 - 45°C for 20 - 40 minutes.

8. A staining method, characterized in that, comprises the following steps: Step 1, performing melanin decolorization on the tissue section according to the melanin decolorization method described in claim 6 or 7; Step 2, staining the tissue section after melanin decolorization.

9. The staining method according to claim 8, characterized in that, the staining is HE staining after decolorization or immunohistochemical staining after decolorization.

Images