Alkaline phosphatase developing solution suitable for full-automatic immunohistochemical staining solution

Abstract

The invention discloses an alkaline phosphatase developing solution suitable for a full-automatic immunohistochemical staining solution, the alkaline phosphatase developing solution comprises a chromogen concentrated solution and a substrate buffer solution, the chromogen concentrated solution comprises naphthol AS-MX phosphate and a Tris buffer solution, and the substrate buffer solution comprises fast red-TR and a Tris buffer solution. According to the invention, the color developing effect shows bright pink, the contrast ratio with blue cell nucleuses after being re-stained by hematoxylin is very obvious, the method is particularly suitable for tumor tissues such as lung cancer and melanoma with pigmentation, the full-automatic staining of tumor tissue slices can be realized, and the core competitiveness of a staining instrument can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and in particular relates to an alkaline phosphatase colorimetric solution suitable for full-automatic immunohistochemical staining solution. Background Art

[0002] DAB color developing solution is the most commonly used color developing reagent in current immunochemical staining technology. In the immune reaction, the horseradish peroxidase carried by the antibody catalyzes the decomposition of the DAB color developing solution, and a yellow-brown colored precipitate is presented on the antigenic site in the tissue section. The coloring precipitate has good stability, is insoluble in various organic solvents, and will not fade even if stored for a long time. It also has a very obvious color difference with the slices after hematoxylin counterstaining, which is convenient for pathologists to diagnose. However, in some special tumor diseases, such as melanoma, lung cancer, etc., the staining effect of the DAB color developing solution and the pigment deposition in the tumor tissue do not have obvious color distinction, which may cause misdiagnosis by the pathologist, thereby affecting the formulation of the treatment plan for the patient. In view of this, it is necessary to study a kind of alkaline phosphatase color developing solution suitable for full-automatic immunohistochemical staining solution. Summary of the invention

[0003] To address the deficiencies of the prior art, the present invention provides an alkaline phosphatase colorimetric solution suitable for fully automatic immunohistochemical staining, comprising a chromogen concentrate and a substrate buffer solution, the coloring effect presenting a bright pink color, which has a very obvious contrast with the blue cell nuclei after hematoxylin counterstaining, and can realize fully automatic staining of tumor tissue sections.

[0004] In order to achieve the above object, the present invention adopts the following technical solution: An alkaline phosphatase colorimetric solution suitable for full-automatic immunohistochemical staining solution comprises a chromogen concentrate and a substrate buffer, wherein the chromogen concentrate comprises naphthol AS-MX phosphate and Tris buffer, and the substrate buffer comprises Fast Red-TR and Tris buffer.

[0005] Preferably, the volume ratio of the chromogen concentrate to the substrate buffer is 1:19.

[0006] Preferably, the mass fraction of the naphthol AS-MX phosphate in the chromogen concentrate is 8%.

[0007] Preferably, the pH of the Tris buffer is 8.5 and the molar concentration is 25 mM.

[0008] Preferably, the mass fraction of Fast Red-TR in the substrate buffer is 1.1%.

[0009] A staining method using alkaline phosphatase colorimetric solution comprises firstly staining a tissue section sample using the alkaline phosphatase colorimetric solution, and then counter-staining the sample using hematoxylin.

[0010] An application of an alkaline phosphatase colorimetric solution is to use the phosphatase colorimetric solution and the DAB colorimetric solution together on a tissue section to achieve staining of two different antibodies.

[0011] The present invention is beneficial in that: (1) The color development effect of the present invention presents a bright pink color, which has a very obvious contrast with the blue cell nucleus after hematoxylin counterstaining. It is particularly suitable for tumor tissues such as lung cancer and melanoma with pigment deposition, and can realize full-automatic staining of tumor tissue sections, which can effectively improve the core competitiveness of the staining instrument; (2) The present invention can be used in conjunction with DAB color developing solution to achieve staining of two different antibodies on a tissue section, effectively saving samples and staining time. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a comparison diagram of the color development effects of different color developing solutions in Example 1 ((A) DAB color developing solution; (B) alkaline phosphatase color developing solution of the present invention); Figure 2 It is a double staining effect test diagram in Example 2 ((A) double staining of P16 (brown) and Ki-67 (red) on cervical cancer tissue section sample; (B) double staining of P63 (brown) and CK pan (red) on breast cancer tissue section sample; (C) double staining of EBER (brown) and CD20 (red) on B cell lymphoma tissue section sample); Figure 3 These are test diagrams of the machine staining effect of the alkaline phosphatase colorimetric solution in Example 3 ((A) staining effect of Calpnion antibody on breast cancer tissue section sample; (B) staining effect of CK pan antibody on breast cancer tissue section sample; (C) staining effect of Ki-67 antibody on tonsil tissue section sample). DETAILED DESCRIPTION

[0013] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0014] An alkaline phosphatase colorimetric solution suitable for fully automatic immunohistochemical staining solution comprises a chromogen concentrate and a substrate buffer at a volume ratio of 1:19, wherein the chromogen concentrate comprises 8% by mass of naphthol AS-MX phosphate and a Tris buffer, and the substrate buffer comprises 1.1% by mass of Fast Red-TR and a Tris buffer, wherein the pH of the Tris buffer is 8.5 and the molar concentration is 25 mM.

[0015] Example 1: Comparison of color development effects of different color developing solutions: Two identical melanoma tissue sections were prepared, and the tissue section samples were stained according to the conventional immunostaining process, wherein one melanoma tissue section was stained using a DAB colorimetric solution, and the other melanoma tissue section was stained using an alkaline phosphatase colorimetric solution of the present invention. After the color development was completed, the above samples were counterstained with hematoxylin, and the color development effects of each colorimetric solution were compared.

[0016] according to Figure 1 It can be seen that the color development effect of the alkaline phosphatase color developing solution of the present invention presents a bright pink color ( Figure 1 (B)), the color difference between the dark brown pigmentation and the dark brown pigmentation is obvious, while the color difference between the brown coloration of the DAB color developing solution and the pigmentation is not obvious ( Figure 1 Therefore, the alkaline phosphatase colorimetric solution of the present invention can replace the DAB colorimetric solution and be used on tumor tissue slice samples with pigment deposition.

[0017] Example 2: DAB color developing solution and alkaline phosphatase color developing solution of the present invention are used in combination: Three different tumor tissue slice samples were prepared, namely, cervical cancer tissue slice samples, breast cancer tissue slice samples, and B-cell lymphoma tissue slice samples. The three samples were stained with antibodies for the first time according to the conventional immunostaining process. After DAB color development was completed, the samples were stained with antibodies for the second time according to the conventional immunostaining process, and then the alkaline phosphatase color development solution of the present invention was used for color development. After the color development was completed, the samples were counterstained with hematoxylin, and the dual color development effect of the combined use of the DAB color development solution and the alkaline phosphatase color development solution of the present invention was observed under a microscope.

[0018] according to Figure 2 It can be seen that P16 antibody and Ki-67 antibody are detected in cervical cancer tissue sections ( Figure 2 (A)), P63 antibody and CKpan antibody in breast cancer tissue sections( Figure 2 (B) ), EBER probe and CD20 antibody in B cell lymphoma tissue sections ( Figure 2 The contrast between the brown coloration and the pink coloration on (C) is clear and bright, which can meet the diagnostic requirements of pathologists. It can be seen that the alkaline phosphatase colorimetric solution of the present invention can be used in combination with the DAB colorimetric solution to achieve staining of two different antibodies on a tissue section, effectively saving samples and staining time.

[0019] Example 3, the dyeing effect of alkaline phosphatase colorimetric solution on the machine: Three different tumor tissue section samples were prepared and stained using alkaline phosphatase colorimetric solution on a fully automatic immunohistochemical stainer CATYS 48 produced by Kaqiu (Jiangsu) Biotechnology Co., Ltd.

[0020] according to Figure 3 It can be seen that the alkaline phosphatase color developing solution produces bright pink color on breast cancer and tonsil tissue sections, which can meet the diagnostic requirements of pathologists. The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. An alkaline phosphatase colorimetric solution suitable for fully automatic immunohistochemical staining solution, characterized in that: The invention comprises a chromogen concentrate and a substrate buffer, wherein the chromogen concentrate comprises naphthol AS-MX phosphate and Tris buffer, and the substrate buffer comprises Fast Red-TR and Tris buffer.

2. The alkaline phosphatase colorimetric solution suitable for fully automatic immunohistochemical staining solution according to claim 1, characterized in that: The volume ratio of the chromogen concentrate to the substrate buffer is 1:

19.

3. The alkaline phosphatase colorimetric solution suitable for fully automatic immunohistochemical staining solution according to claim 1, characterized in that: The mass fraction of the naphthol AS-MX phosphate in the chromogen concentrate is 8%.

4. The alkaline phosphatase colorimetric solution suitable for fully automatic immunohistochemical staining solution according to claim 1, characterized in that: The pH of the Tris buffer is 8.5, and the molar concentration is 25 mM.

5. The alkaline phosphatase colorimetric solution suitable for fully automatic immunohistochemical staining solution according to claim 1, characterized in that: The mass fraction of the Fast Red-TR in the substrate buffer solution is 1.1%.

6. The method for dyeing an alkaline phosphatase color developing solution according to claim 1, characterized in that: The tissue sections were first stained with alkaline phosphatase colorimetric solution and then counterstained with hematoxylin.

7. The use of an alkaline phosphatase color developing solution according to claim 1, characterized in that: Phosphatase colorimetric solution and DAB colorimetric solution were used together on a tissue section to achieve staining with two different antibodies.

Images