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

Abstract

The application discloses an alkaline phosphatase color developing solution suitable for a full-automatic immunohistochemical staining instrument, which comprises a chromogen concentrated solution and a substrate buffer solution, wherein 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. The color developing effect of the alkaline phosphatase color developing solution presents bright pink, and the contrast with the blue cell nucleus after hematoxylin counterstaining is very obvious, and the alkaline phosphatase color developing solution is particularly suitable for tumor tissues such as lung cancer and melanoma with pigment deposition, can realize full-automatic staining of tumor tissue sections, and can effectively improve the core competitiveness of the staining instrument.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to an alkaline phosphatase staining solution suitable for fully automated immunohistochemical staining instruments. Background Technology

[0002] DAB chromogenic solution is currently the most commonly used chromogenic reagent in immunochemical staining techniques. During the immunoreaction, horseradish peroxidase carried by the antibody catalyzes the decomposition of DAB chromogenic solution, resulting in a yellow-brown stained precipitate at the antigen sites in the tissue section. This stained precipitate is stable, insoluble in various organic solvents, does not fade during long-term storage, and exhibits a very clear color difference from sections counterstained with hematoxylin, facilitating diagnosis by pathologists. However, in some specific tumor diseases, such as melanoma and lung cancer, the staining effect of DAB chromogenic solution is not clearly distinguishable from the pigment deposition in tumor tissue, which may lead to misdiagnosis by pathologists and thus affect the patient's treatment plan. Therefore, it is necessary to research an alkaline phosphatase chromogenic solution suitable for fully automated immunohistochemical staining instruments. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide an alkaline phosphatase staining solution suitable for fully automated immunohistochemical staining instruments, comprising a chromogen concentrate and a substrate buffer. The staining effect produces a bright pink color with a significant contrast to the blue cell nuclei after hematoxylin counterstaining, enabling fully automated staining of tumor tissue sections.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] An alkaline phosphatase chromogenic solution suitable for a fully automated immunohistochemical staining instrument, comprising a chromogen concentrate and a substrate buffer, wherein the chromogen concentrate comprises naphthol AS-MX phosphate and Tris buffer, and the substrate buffer comprises Solid Red-TR and Tris buffer.

[0006] Preferably, the volume ratio of the chromogen concentrate to the substrate buffer is 1:19.

[0007] Preferably, the mass fraction of the naphthol AS-MX phosphate in the colorant concentrate is 8%.

[0008] Preferably, the Tris buffer solution has a pH of 8.5 and a molar concentration of 25 mM.

[0009] Preferably, the mass fraction of the solid red-TR in the substrate buffer is 1.1%.

[0010] A staining method using alkaline phosphatase chromogenic solution involves first staining tissue sections with the alkaline phosphatase chromogenic solution, and then counterstaining the samples with hematoxylin.

[0011] An application of an alkaline phosphatase chromogenic solution, which combines phosphatase chromogenic solution and DAB chromogenic solution on a single tissue section to achieve staining with two different antibodies.

[0012] The advantages of this invention are:

[0013] (1) The color development effect of the present invention presents a bright pink color, which is very obvious in contrast to the blue cell nuclei after hematoxylin counterstaining. It is particularly suitable for tumor tissues such as lung cancer and melanoma with pigment deposition. It can realize fully automatic staining of tumor tissue sections and effectively enhance the core competitiveness of the staining instrument.

[0014] (2) This invention can be used in conjunction with DAB staining solution to achieve staining of two different antibodies on a single tissue section, effectively saving sample and staining time. Attached Figure Description

[0015] Figure 1 These are comparison diagrams of the color development effects of different colorimetric solutions in Example 1 ((A) DAB colorimetric solution; (B) alkaline phosphatase colorimetric solution of the present invention).

[0016] Figure 2 These are the double staining effect test images from Example 2 ((A) double staining of P16 (brown) and Ki-67 (red) on cervical cancer tissue samples; (B) double staining of P63 (brown) and CK pan (red) on breast cancer tissue samples; (C) double staining of EBER (brown) and CD20 (red) on B-cell lymphoma tissue samples).

[0017] Figure 3 These are the staining effect test images of the alkaline phosphatase staining 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 Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0019] An alkaline phosphatase chromogenic solution suitable for a fully automated immunohistochemical staining instrument includes a chromogen concentrate and a substrate buffer at a volume ratio of 1:19. The chromogen concentrate includes 8% naphthol AS-MX phosphate and Tris buffer, and the substrate buffer includes 1.1% solid red-TR and Tris buffer. The Tris buffer has a pH of 8.5 and a molar concentration of 25 mM.

[0020] Example 1: Comparison of color development effects of different color developing solutions:

[0021] Two identical melanoma tissue sections were prepared and stained according to standard immunostaining procedures. One melanoma tissue section was stained using DAB chromogenic solution, while the other melanoma tissue section was stained using the alkaline phosphatase chromogenic solution of this invention. After staining, the samples were counterstained with hematoxylin, and the staining effects of each chromogenic solution were compared.

[0022] according to Figure 1 It can be seen that the alkaline phosphatase colorimetric solution of the present invention exhibits a bright pink color development effect. Figure 1 (B) shows a clear color difference with the dark brown pigment deposition, while the brown color of the DAB developing solution does not show a clear color difference with the pigment deposition. Figure 1 (A) Therefore, the alkaline phosphatase chromogenic solution of the present invention can replace the DAB chromogenic solution for use on tumor tissue section samples with pigment deposition.

[0023] Example 2: Combined use of DAB chromogenic solution and the alkaline phosphatase chromogenic solution of the present invention:

[0024] Three different tumor tissue samples were prepared: cervical cancer tissue samples, breast cancer tissue samples, and B-cell lymphoma tissue samples. Each sample underwent a first antibody staining according to standard immunostaining procedures. After DAB staining was completed, a second antibody staining was performed according to standard immunostaining procedures, followed by staining with the alkaline phosphatase chromogenic solution of this invention. After staining was completed, the samples were counterstained with hematoxylin, and the dual staining effect of using the combined DAB chromogenic solution and the alkaline phosphatase chromogenic solution of this invention was observed under a microscope.

[0025] according to Figure 2 It can be seen that P16 antibody and Ki-67 antibody are present 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 2The contrast between the brown and pink staining on (C) is clear and the colors are vibrant, meeting the diagnostic requirements of pathologists. Therefore, the alkaline phosphatase staining solution of this invention can be used in conjunction with DAB staining solution to achieve staining with two different antibodies on a single tissue section, effectively saving sample and staining time.

[0026] Example 3: Staining effect of alkaline phosphatase chromogenic solution on machine:

[0027] Three different tumor tissue sections were prepared and stained using alkaline phosphatase staining solution on a CATYS 48 fully automated immunohistochemical staining instrument manufactured by CATYS (Jiangsu) Biotechnology Co., Ltd.

[0028] according to Figure 3 It is known that the alkaline phosphatase staining solution produces a bright pink color on breast cancer and tonsil tissue sections, which can meet the diagnostic requirements of pathologists.

[0029] The foregoing has shown and described 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 way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A staining method suitable for a fully automated immunohistochemical stainer, characterized in that, The method comprises the following steps: The tumor tissue section sample is dyed, color development is performed using an alkaline phosphatase color developing solution, after color development is completed, the sample is re-dyed using hematoxylin, and the color development effect is pink, and the contrast of the blue cell nucleus after re-dyeing with hematoxylin is obvious; The alkaline phosphatase color developing solution comprises a chromogen concentrate and a substrate buffer solution, the chromogen concentrate comprises naphthol AS-MX phosphate and a Tris buffer solution, the substrate buffer solution comprises fast red-TR and a Tris buffer solution, the volume ratio of the chromogen concentrate to the substrate buffer solution is 1:19, the mass fraction of the naphthol AS-MX phosphate in the chromogen concentrate is 8%, the Tris buffer solution has a pH of 8.5 and a molar concentration of 25 mM, and the mass fraction of the fast red-TR in the substrate buffer solution is 1.1%.

2. The staining method suitable for a fully automated immunohistochemical stainer according to claim 1, characterized in that, The alkaline phosphatase color developing solution and the DAB color developing solution are combined for use on one tissue section, so that the staining of two different antibodies is realized.

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