技术领域Technical field
本发明属于免疫组化技术领域，具体涉及一种双重免疫组化染色试剂盒及其在鉴别良恶性胆管上皮肿瘤中的应用。The invention belongs to the technical field of immunohistochemistry, and specifically relates to a double immunohistochemical staining kit and its application in identifying benign and malignant bile duct epithelial tumors.
背景技术Background technique
免疫组织化学（Immunohistochemisty，IHC）又称免疫细胞化学，简称免疫组化，是病理学诊断、鉴别诊断，肿瘤特异性分子靶标筛选、鉴定的最重要平台性技术。常规的免疫组化是应用免疫学基本原理——抗原抗体反应，即抗原与抗体特异性结合的原理，通过化学反应使标记抗体的显色剂（荧光素、酶、金属离子、同位素）显色来确定组织细胞内抗原（多肽和蛋白质），对其进行定位、定性及相对定量的研究。一般是一张切片观察一个抗原的染色结果。多色免疫组化能在一张切片上观察多个抗原的染色结果，不同抗原之间的关系对比直接，有利于节省组织与肿瘤微浸润的观察。目前，多色免疫组化技术已经应用到前列腺、乳腺、消化道等多种疾病的病理诊断中，技术难点在于找到合适的两种或多种合适的标记物以使用一抗和二抗共同标记在同一组织切片上，以达到颜色对比鲜明，定位准确的效果。Immunohistochemistry (IHC), also known as immunocytochemistry, or immunohistochemistry for short, is the most important platform technology for pathological diagnosis, differential diagnosis, and screening and identification of tumor-specific molecular targets. Conventional immunohistochemistry is the application of the basic principles of immunology - antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody. The chromogenic reagent (fluorescein, enzyme, metal ion, isotope) labeled with the antibody is colored through a chemical reaction. To determine the antigens (polypeptides and proteins) in tissue cells, conduct localization, qualitative and relative quantitative research on them. Generally, one slice is used to observe the staining results of one antigen. Multicolor immunohistochemistry can observe the staining results of multiple antigens on one section. The relationship between different antigens is directly compared, which is beneficial to saving tissue and observing tumor micro-infiltration. At present, multi-color immunohistochemistry technology has been applied to the pathological diagnosis of prostate, breast, digestive tract and other diseases. The technical difficulty lies in finding two or more suitable markers to co-label with primary and secondary antibodies. On the same tissue section, to achieve sharp color contrast and accurate positioning.
肝内胆管癌（ICC）约占原发性肝癌的10%～20%，占胆管癌的6%～20%，是发生率仅次于肝细胞癌（HCC）的第二常见肝脏恶性肿瘤。ICC具有高度恶性的生物学行为，目前ICC患者的术后5年生存率一般在30%～50%之间。已知许多致病因素与ICC的发生有密切关系，包括华支睾吸虫、肝内胆管结石、慢性胆管炎、HBV/HCV感染，原发性硬化性胆管炎、寄生虫感染、胆管畸形（胆总管囊肿和Caroli病）、肝硬化、先天性肝纤维化等。对于这些具有慢性肝损伤的基础疾病的患者，包括胆汁淤积性肝损伤、肝内和/或肝外胆道狭窄以及其他胆道异常的患者，由于炎症的反复刺激，可引起细胆管的反应性增生，亦可进一步恶变，继发ICC或HCC。Intrahepatic cholangiocarcinoma (ICC) accounts for approximately 10% to 20% of primary liver cancers and 6% to 20% of cholangiocarcinomas. It is the second most common liver malignant tumor after hepatocellular carcinoma (HCC). ICC has highly malignant biological behavior. Currently, the 5-year postoperative survival rate of ICC patients is generally between 30% and 50%. Many pathogenic factors are known to be closely related to the occurrence of ICC, including Clonorchis sinensis, intrahepatic bile duct stones, chronic cholangitis, HBV/HCV infection, primary sclerosing cholangitis, parasitic infection, bile duct malformation (biliary malformation) Common duct cyst and Caroli disease), cirrhosis, congenital liver fibrosis, etc. For these patients with underlying diseases of chronic liver injury, including cholestatic liver injury, intrahepatic and/or extrahepatic biliary stenosis, and other biliary abnormalities, repeated stimulation of inflammation can cause reactive proliferation of small bile ducts. It may also undergo further malignant transformation, leading to secondary ICC or HCC.
细胆管反应是界板门管区的反应性病变，包括细胆管增生，伴有间质纤维化及炎细胞浸润，可见于晚期慢性肝病（如慢性病毒性肝炎），也见于晚期慢性肝病中出现的HCC和ICC等肝肿瘤周围。当ICC的细胞异型性不明显时，ICC的组织学与胆管反应极其相似，且ICC通常表现为浸润替代生长模式，有时很难对ICC和旺炽性胆管增生进行鉴别。Bile duct reaction is a reactive lesion in the portal duct area of the plate, including bile duct proliferation, accompanied by interstitial fibrosis and inflammatory cell infiltration. It can be seen in advanced chronic liver diseases (such as chronic viral hepatitis) and also in HCC that appears in advanced chronic liver diseases. and around liver tumors such as ICC. When the cellular atypia of ICC is not obvious, the histology of ICC is very similar to bile duct reaction, and ICC usually shows an infiltrative alternative growth pattern, making it sometimes difficult to differentiate between ICC and vigorous bile duct hyperplasia.
胆管腺瘤是一种良性肿瘤或瘤样增生性病变，约82.9%为单发，少数可以多发。大小通常<1cm，多位于肝包膜下，也可存在肝实质深部，大体呈白色或灰白色，圆形或卵圆形，质地较硬，边界清晰，无真性包膜。镜下由大小较一致的小胆管聚集构成，管腔狭小，弯曲状或实性条索状，多不含浓缩胆汁，类似于胆管反应，可能是ICC的前期病变。目前为止，胆管腺瘤的生物学性质尚未完全阐明，且胆管腺瘤与ICC的区别诊断有时也是极其困难。Bile duct adenoma is a benign tumor or tumor-like proliferative lesion. About 82.9% of the tumors are solitary, and a few may have multiple tumors. The size is usually <1cm, mostly located under the liver capsule, and may also be found deep in the liver parenchyma. They are generally white or off-white, round or oval, with a hard texture, clear boundaries, and no true capsule. Microscopically, it is composed of a collection of small bile ducts of relatively uniform size. The lumens are narrow, curved or solid, and mostly do not contain concentrated bile. They are similar to bile duct reactions and may be early lesions of ICC. So far, the biological properties of bile duct adenomas have not been fully elucidated, and the differential diagnosis of bile duct adenomas and ICC is sometimes extremely difficult.
新版WHO肿瘤（2019年版）消化系统肿瘤分类标准(WHO Classification ofTumours Editorial Board: Digestive System Tumours, 5thedition, 2019，vol.1；Http://pubications.iarc.fr/529)中的ICC的特殊亚型细胆管癌（CLC），特指起源于细胆管或Hering管的腺癌，组织学上有时可类似小胆管或反应性小胆管增生。CLC癌细胞呈小立方形，细胞核/浆比增高，核卵圆形，胞浆淡染，缺乏黏液，异型性小，呈高分化状态，在组织学上排列成较规则的成角小导管、裂隙状或条索状，以在透明变性的胶原纤维间质内呈稀松的分枝状或“鹿角状”排列为特征。对于高分化CLC有时需要与胆管错构瘤及胆管腺瘤相鉴别。Special features of ICC in the new WHO Classification of Tumors (2019 edition) Digestive System Tumors (WHO Classification of Tumours Editorial Board: Digestive System Tumours,5th edition, 2019, vol.1; http://publications.iarc.fr/529) Subtype cholangiocarcinoma (CLC) refers specifically to adenocarcinoma originating from small bile ducts or Hering's ducts. Histologically, it can sometimes resemble small bile ducts or reactive small bile duct hyperplasia. CLC cancer cells are small cuboidal in shape, with an increased nucleus/plasma ratio, oval nuclei, light staining of the cytoplasm, lack of mucus, small atypia, and a well-differentiated state. Histologically, they are arranged into more regular angular small ducts, Slit-like or cord-like, characterized by a loose branch-like or "antler-like" arrangement within the hyaline-denatured collagen fiber matrix. Well-differentiated CLC sometimes needs to be differentiated from bile duct hamartomas and bile duct adenomas.
炎症引起的反应性旺炽性增生、胆管错构瘤、胆管板畸形、胆管腺瘤或是高分化ICC，有时诊断可能极具挑战性。而在肝脏恶性肿瘤中有时会同时存在的胆管上皮，这些胆管上皮到底是肿瘤性的还是反应的，是混合性HCC-ICC，还是伴胆管反应的HCC，鉴别这些对恶性肿瘤的准确分型及正确诊断同样至关重要。因此，本领域极其需要免疫组织化学染色等其他工具来提高诊断灵敏度和准确性。Diagnosis can sometimes be challenging due to inflammation-induced reactive hyperplasia, bile duct hamartomas, bile duct plate malformations, bile duct adenomas, or well-differentiated ICC. In liver malignant tumors, bile duct epithelium sometimes coexists. Whether these bile duct epithelium are neoplastic or reactive, mixed HCC-ICC, or HCC with bile duct reaction, how to identify these accurate classifications of malignant tumors and Proper diagnosis is also crucial. Therefore, there is a great need in the field for additional tools such as immunohistochemical staining to improve diagnostic sensitivity and accuracy.
果蝇zeste基因增强子的人类同源物 2(Enhancer of zeste homolog 2，EZH2)具有DNA甲基转移和组蛋白脱乙酰化酶的调控作用，可参与调控癌症进展和干细胞的多潜能性等生命活动。研究发现，其在ICC中表达明显升高。Sasaki等发现，EZH2在全部CLC高表达，在胆管腺瘤和反应性小胆管增生中不表达（Am JSurg Pathol 2014;38：364)。因此，EZH2的免疫组化染色可用于鉴别ICC（阳性）、胆管反应旺炽性增生、胆管错构瘤、胆管板畸形、胆管腺瘤（阴性）。CK7(细胞角蛋白7 )为角蛋白基因家族的一员，可用于勾勒出胆上皮（PatholRes Pract 2003;199：65)。The human homolog 2 (Enhancer of zeste homolog 2, EZH2) of the Drosophilazeste gene has the regulatory role of DNA methyl transfer and histone deacetylase, and can participate in the regulation of cancer progression and the pluripotency of stem cells. Activity. Studies have found that its expression in ICC is significantly elevated. Sasaki et al. found that EZH2 is highly expressed in all CLC and is not expressed in bile duct adenomas and reactive small bile duct hyperplasia (Am JSurg Pathol 2014;38:364). Therefore, immunohistochemical staining of EZH2 can be used to identify ICC (positive), bile duct hyperplasia, bile duct hamartoma, bile duct plate malformation, and bile duct adenoma (negative). CK7 (cytokeratin 7) is a member of the keratin gene family and can be used to outline the biliary epithelium (PatholRes Pract 2003;199:65).
目前，在日常病理诊断中，经常需要做多种免疫组化检测。但由于技术的限制，常用的免疫组化方法是在1张病理组织玻片上用一种颜色（一般为棕色）标记一种蛋白。当分析多种蛋白指标时，需要用对应的抗体分别在多张不同的玻片上标记。这样的单一标记的免疫组化染色方法，缺点如下：①每一个标记需要一张组织切片，标本浪费；②分别染色在两个玻片上的两个免疫组化指标是否同时表达于同一群细胞，靠经验和推测，需要反复对比HE及免疫组化切片，阅片难度大。没有经验的医师往往将恶性肿瘤中轻度异性增生的胆管上皮误认为恶性，从而导致误诊。At present, in daily pathological diagnosis, a variety of immunohistochemical tests are often required. However, due to technical limitations, the commonly used immunohistochemical method is to label a protein with a color (usually brown) on a pathological tissue slide. When analyzing multiple protein indicators, it is necessary to label multiple different slides with corresponding antibodies. The disadvantages of such a single-marker immunohistochemical staining method are as follows: ① Each marker requires a tissue section, which wastes the specimen; ② Whether the two immunohistochemical indicators stained on two slides are expressed simultaneously in the same group of cells, Relying on experience and speculation, it is necessary to repeatedly compare HE and immunohistochemistry slices, making it difficult to read the slices. Inexperienced doctors often mistake mild dysplasia of bile duct epithelium in malignant tumors for malignancy, leading to misdiagnosis.
发明内容Contents of the invention
本发明要解决的技术问题是良恶性胆管上皮肿瘤难以快速准确判断的技术问题。The technical problem to be solved by the present invention is that it is difficult to quickly and accurately judge benign and malignant bile duct epithelial tumors.
本发明首先提供了一种双重免疫组化染色试剂盒。该试剂盒包括一抗试剂、二抗试剂 和染色剂；所述一抗试剂包括有EZH2抗体和CK7抗体；所述二抗试剂包括组合二抗（HRP标记抗小鼠IgG/HRP+兔抗IgG/AP）或组合二抗（HRP标记抗小鼠IgG/AP+兔抗IgG/HRP），所述二抗试剂包括EZH2二抗和 CK7二抗。The present invention first provides a double immunohistochemical staining kit. The kit includes primary antibody reagents, secondary antibody reagents and staining agents; the primary antibody reagents include EZH2 antibodies and CK7 antibodies; the secondary antibody reagents include combined secondary antibodies (HRP labeled anti-mouse IgG/HRP+rabbit anti-IgG/ AP) or combined secondary antibody (HRP-labeled anti-mouse IgG/AP + rabbit anti-IgG/HRP). The secondary antibody reagents include EZH2 secondary antibody and CK7 secondary antibody.
其中，所述EZH2抗体和CK7抗体来源于两种不同种属的动物。进一步的，两种不同种属的动物分别为鼠或兔。Wherein, the EZH2 antibody and CK7 antibody are derived from two different species of animals. Further, the two different species of animals are rats or rabbits.
其中，所述染色剂为过氧化物酶HRP 辣根介导的染色剂和碱性磷酸酶AP介导的染色剂。Wherein, the staining agent is a peroxidase HRP horseradish-mediated staining agent and an alkaline phosphatase AP-mediated staining agent.
其中，所述一抗试剂和二抗试剂中的针对同一抗原的抗体来源相匹配。进一步的，所述EZH2抗体来源于鼠或兔，CK7抗体来源于兔或鼠，EZH2抗体和CK7抗体来源动物不同。Wherein, the sources of antibodies directed against the same antigen in the primary antibody reagent and the secondary antibody reagent match. Further, the EZH2 antibody is derived from mice or rabbits, the CK7 antibody is derived from rabbits or mice, and the EZH2 antibodies and CK7 antibodies are derived from different animals.
进一步的，上述二抗试剂含有与上述一抗试剂中的EZH2或CK7抗体种属来源相匹配的辣根过氧化物酶（HRP）标记的二抗聚合物；上述二抗试剂含有与上述一抗试剂中的EZH2或CK7抗体种属来源相匹配的碱性磷酸酶（AP）标记的二抗聚合物；所述二抗试剂中的EZH2二抗和CK7二抗的标记不同。Further, the above-mentioned secondary antibody reagent contains a horseradish peroxidase (HRP)-labeled secondary antibody polymer that matches the species source of the EZH2 or CK7 antibody in the above-mentioned primary antibody reagent; the above-mentioned secondary antibody reagent contains a horseradish peroxidase (HRP)-labeled secondary antibody polymer that matches the species source of the above-mentioned primary antibody. The EZH2 or CK7 antibody in the reagent is an alkaline phosphatase (AP)-labeled secondary antibody polymer that matches the species source; the EZH2 secondary antibody and CK7 secondary antibody in the secondary antibody reagent are labeled differently.
比如，上述二抗试剂包括组合二抗（HRP标记抗小鼠IgG/HRP+兔抗IgG/AP）或组合二抗（HRP标记抗小鼠IgG/AP+兔抗IgG/HRP）。For example, the above-mentioned secondary antibody reagents include combined secondary antibodies (HRP-labeled anti-mouse IgG/HRP + rabbit anti-IgG/AP) or combined secondary antibodies (HRP-labeled anti-mouse IgG/AP + rabbit anti-IgG/HRP).
其中，上述双重免疫组化染色试剂盒中还包括染色剂，所述染色剂包括辣根过氧化物酶底物染色液和碱性磷酸酶底物染色液。Wherein, the above-mentioned double immunohistochemical staining kit also includes a staining agent, and the staining agent includes a horseradish peroxidase substrate staining solution and an alkaline phosphatase substrate staining solution.
其中，上述辣根过氧化物酶底物染色液包括3,3-二氨基联苯胺盐酸盐（DAB）；或者所述碱性磷酸酶底物染色液包括红色碱性磷酸酶底物（具体可以为Fast Red A和Fast RedB）。Wherein, the horseradish peroxidase substrate dyeing solution includes 3,3-diaminobenzidine hydrochloride (DAB); or the alkaline phosphatase substrate dyeing solution includes red alkaline phosphatase substrate (specifically Can be Fast Red A and Fast RedB).
进一步的，上述染色剂还包括复染染色液，所述复染染色液的显色不掩盖所述辣根过氧化物酶底物染色液和碱性磷酸酶底物染色液的显色。Furthermore, the above-mentioned dyeing agent also includes a counterstain dyeing solution, and the color development of the counterstaining dyeing solution does not mask the color development of the horseradish peroxidase substrate dyeing solution and alkaline phosphatase substrate dyeing solution.
其中，上述的双染免疫组化试剂盒中还可以含有抗原修复剂和/内源性过氧化氢酶封闭试剂。Among them, the above-mentioned double-staining immunohistochemistry kit may also contain an antigen retrieval agent and/or an endogenous catalase blocking reagent.
进一步的，上述双重免疫组化染色试剂盒用于对组织切片进行染色。进一步的，所述的组织切片为石蜡组织切片和冰冻组织切片。Further, the above-mentioned double immunohistochemical staining kit was used to stain tissue sections. Further, the tissue sections are paraffin tissue sections and frozen tissue sections.
可以理解的是上述试剂盒中的各种试剂为独立包装。It can be understood that various reagents in the above kit are individually packaged.
本发明还进一步地提供了上述所述的双重免疫组化染色试剂盒在制备鉴别良恶性胆管上皮肿瘤的试剂中的应用。The present invention further provides the application of the above-mentioned double immunohistochemical staining kit in preparing reagents for identifying benign and malignant bile duct epithelial tumors.
本发明还在上述方案基础上提供了一种肝脏组织切片进行双重免疫组化染色的方法。该方法其特征在于包括以下步骤三种操作方式：The present invention also provides a method for double immunohistochemical staining of liver tissue sections based on the above scheme. The method is characterized by three operating modes including the following steps:
A：A:
(1) 准备组织切片，进行抗原修复、内源性过氧化氢酶封闭；(1) Prepare tissue sections for antigen retrieval and endogenous catalase blocking;
(2)使用一抗试剂对组织切片进行孵育：采用鸡尾酒一抗，该鸡尾酒一抗包含鼠来源EZH2一抗和兔来源CK7一抗，或者包括兔来源EZH2一抗和鼠来源CK7一抗；(2) Use primary antibody reagents to incubate tissue sections: use a cocktail primary antibody, which contains a mouse-derived EZH2 primary antibody and a rabbit-derived CK7 primary antibody, or a rabbit-derived EZH2 primary antibody and a mouse-derived CK7 primary antibody;
（3）二抗孵育与显色反应：先使用与EZH2或CK7抗体种属来源相匹配的辣根过氧化物酶（HRP）标记二抗进行孵育，介导3,3-二氨基联苯胺盐酸盐（DAB）反应，产生不溶于水和乙醇的棕黄色或棕褐色沉淀；再进行与剩余的CK7或EZH2抗体种属来源相匹配的碱性磷酸酶（AP）标记二抗进行孵育介导红色底物显色系统染色；(3) Secondary antibody incubation and color development reaction: First use horseradish peroxidase (HRP)-labeled secondary antibody that matches the species source of the EZH2 or CK7 antibody for incubation, mediating 3,3-diaminobenzidine salt Acid acid (DAB) reaction produces a brown or tan precipitate that is insoluble in water and ethanol; then incubation is mediated with an alkaline phosphatase (AP)-labeled secondary antibody that matches the species source of the remaining CK7 or EZH2 antibodies. Red substrate chromogenic system staining;
（4）切片复染；保存备用。(4) Counterstain the sections; save for later use.
根据酶标二抗不同可呈现不同的结果：Different results can be presented depending on the enzyme-labeled secondary antibody:
HRP标记二抗与EZH2结合/AP标记二抗与CK7结合：EZH2棕色核染色和CK7红色胞浆染色；HRP-labeled secondary antibody binds to EZH2/AP-labeled secondary antibody binds to CK7: EZH2 brown nuclear staining and CK7 red cytoplasmic staining;
HRP标记二抗与CK7结合/AP标记二抗与EZH2结合：CK7棕色胞浆染色和EZH2红色核染色。HRP-labeled secondary antibody binds to CK7/AP-labeled secondary antibody binds to EZH2: CK7 brown cytoplasmic staining and EZH2 red nuclear staining.
或者B：Or B:
（1）准备组织切片，进行抗原修复、内源性过氧化氢酶封闭；(1) Prepare tissue sections for antigen retrieval and endogenous catalase blocking;
（2）使用一抗试剂对组织切片进行孵育：采用鸡尾酒一抗，该鸡尾酒一抗包含鼠来源EZH2一抗和兔来源CK7一抗，或者包括兔来源EZH2一抗和鼠来源CK7一抗；(2) Use primary antibody reagents to incubate tissue sections: use a cocktail primary antibody, which contains a mouse-derived EZH2 primary antibody and a rabbit-derived CK7 primary antibody, or a rabbit-derived EZH2 primary antibody and a mouse-derived CK7 primary antibody;
（3）二抗孵育与显色反应：采用鸡尾酒二抗，同时介导酶促显色反应；该鸡尾酒二抗包含分别针对EZH2或CK7的辣根过氧化物酶（HRP）标记二抗和碱性磷酸酶（AP）标记二抗，并与对应的一抗的种属来源相匹配； HRP介导生成棕黄色或棕褐色沉淀，AP介导生成红色沉淀；(3) Secondary antibody incubation and color reaction: Use a cocktail secondary antibody to mediate an enzymatic color reaction at the same time; the cocktail secondary antibody contains horseradish peroxidase (HRP)-labeled secondary antibody and alkali targeting EZH2 or CK7 respectively. The secondary antibody is labeled with phosphatase (AP) and matches the species source of the corresponding primary antibody; HRP mediates the generation of brown or tan precipitate, and AP mediates the generation of red precipitate;
（4）切片复染；保存备用。(4) Counterstain the sections; save for later use.
根据酶标二抗不同可呈现不同的结果：Different results can be presented depending on the enzyme-labeled secondary antibody:
HRP标记二抗与EZH2结合/AP标记二抗与CK7结合：EZH2棕色核染色和CK7红色胞浆染色；HRP-labeled secondary antibody binds to EZH2/AP-labeled secondary antibody binds to CK7: EZH2 brown nuclear staining and CK7 red cytoplasmic staining;
HRP标记二抗与CK7结合/AP标记二抗与EZH2结合：CK7棕色胞浆染色和EZH2红色核染色。HRP-labeled secondary antibody binds to CK7/AP-labeled secondary antibody binds to EZH2: CK7 brown cytoplasmic staining and EZH2 red nuclear staining.
或者C：Or C:
（1）准备组织切片，进行抗原修复、内源性过氧化氢酶封闭；(1) Prepare tissue sections for antigen retrieval and endogenous catalase blocking;
（2）使用鼠或兔来源EZH2或CK7一抗孵育，使用与一抗种属来源相匹配的辣根过氧化物酶（HRP）标记二抗进行孵育，介导3,3-二氨基联苯胺盐酸盐（DAB）反应，产生不溶于水和乙醇的棕黄色或棕褐色沉淀；(2) Incubate with EZH2 or CK7 primary antibody derived from mouse or rabbit, and incubate with horseradish peroxidase (HRP)-labeled secondary antibody that matches the species source of the primary antibody to mediate 3,3-diaminobenzidine. Hydrochloride (DAB) reaction produces a brown or tan precipitate that is insoluble in water and ethanol;
（3）二抗孵育与显色反应：然后与用前述一抗来源不同且针对的抗原不同的，鼠或兔来源的CK7或EZH2的一抗进行孵育；孵育后用与一抗种属来源相同的碱性磷酸酶（AP）标记二抗进行孵育，介导基于AP催化的生成红色沉淀；(3) Secondary antibody incubation and color development reaction: Then incubate with the primary antibody of CK7 or EZH2 from mouse or rabbit, which is from a different source and targets a different antigen than the aforementioned primary antibody; after incubation, use the same species source as the primary antibody Alkaline phosphatase (AP)-labeled secondary antibody is incubated to mediate the generation of red precipitate based on AP catalysis;
（4）复染；保存备用。(4) Counterstain; save for later use.
根据一抗选择先后顺序呈现不同的结果：Different results will be presented depending on the order of primary antibody selection:
步骤一选择EZH2/步骤二选择CK7：EZH2棕色核染色和CK7红色胞浆染色；Select EZH2 in Step 1/Select CK7 in Step 2: EZH2 brown nuclear staining and CK7 red cytoplasmic staining;
步骤一选择CK7/步骤二选择EZH2：CK7棕色胞浆染色和EZH2红色核染色。Select CK7 in Step 1/Select EZH2 in Step 2: CK7 brown cytoplasmic staining and EZH2 red nuclear staining.
使用该方法制备的双重染色切片，可用于进行显微观察，以鉴别良恶性胆管上皮肿瘤。Double-stained sections prepared using this method can be used for microscopic observation to identify benign and malignant bile duct epithelial tumors.
本发明的有益效果：本发明技术方案利用双重免疫组化技术，针对两个有关联抗原蛋白指标CK7和EZH2，在一张切片上能同时勾勒出反应性胆管与癌变的胆管上皮，且能观察到两者之间的关系，能帮助识别肿瘤与非肿瘤性胆管，从而有助于正确的病理诊断。利用本发明技术方案，使得同时标记的两个抗原指标是否同时表达于同一群细胞变得所见即所得，直接以显微镜下观察的结果做判断，而不再靠经验和推测做判断，降低了阅片难度，提高了工作效率及判断准确性，达到了“1+1>2”的效果。另一方面，由于一张玻片可以同时检测两个蛋白指标，也减少了组织切片的使用量，具有好的技术效果。本发明技术方案经的临床实验，证实在鉴别良恶性胆管上皮肿瘤细胞系别、分型和恶性程度判断提高诊断灵敏度和准确性有较好的应用前景。Beneficial effects of the present invention: The technical solution of the present invention uses dual immunohistochemistry technology to target two related antigen protein indicators CK7 and EZH2, and can simultaneously outline reactive bile ducts and cancerous bile duct epithelium on one slice, and can observe Understanding the relationship between the two can help identify tumor and non-tumor bile ducts, thereby contributing to correct pathological diagnosis. Utilizing the technical solution of the present invention, whether two simultaneously labeled antigen indicators are simultaneously expressed in the same group of cells becomes WYSIWYG, and the judgment is made directly based on the results of observation under a microscope instead of relying on experience and speculation, which reduces the risk of The difficulty of reading pictures improves work efficiency and accuracy of judgment, achieving the effect of "1+1>2". On the other hand, since one slide can detect two protein indicators at the same time, it also reduces the use of tissue sections and has good technical effects. Clinical experiments of the technical solution of the present invention have confirmed that it has good application prospects in identifying benign and malignant bile duct epithelial tumor cell lines, typing and malignant degree judgment to improve diagnostic sensitivity and accuracy.
附图说明Description of drawings
图1、病例1肝包块HE低倍照片 （4× ）。Figure 1. HE low-magnification photo of the liver mass in Case 1 (4×).
图2、病例1肝包块HE中倍照片（10× ）。Figure 2. HE medium-magnification photo of the liver mass in Case 1 (10×).
图3、病例1肝包块双染免疫组化低倍照片（4× ）。Figure 3. Double-stained immunohistochemistry low-magnification photo of the liver mass in Case 1 (4×).
图4、病例1肝包块双染免疫组化中倍照片（10× ）。Figure 4. Double-stained immunohistochemistry medium-magnification photo of the liver mass in Case 1 (10×).
图5、病例2肝占位HE中倍照片（10× ）。Figure 5. HE medium-magnification photo of liver occupancy in Case 2 (10×).
图6、病例2肝占位单染的CK7免疫组化中倍照片（10× ）。Figure 6. Medium-magnification photo (10×) of single-stained CK7 immunohistochemistry of case 2 liver.
图7、病例2肝占位单染的EZH2免疫组化中倍照片（10× ）。Figure 7. Medium-magnification photo (10×) of single-stained EZH2 immunohistochemistry in the liver of case 2.
图8、病例2肝占位双重染色免疫组化中高倍照片（20× ）。Figure 8. Medium-high magnification photo of case 2 liver mass double staining immunohistochemistry (20×).
图9、病例3肝包块HE低倍照片 （4× ）。Figure 9. HE low-magnification photo of the liver mass in Case 3 (4×).
图10、病例3肝包块HE中倍照片（10× ）。Figure 10. HE medium-magnification photo of the liver mass in Case 3 (10×).
图11、病例3肝包块双染免疫组化低倍照片（4× ）。Figure 11. Double-stained immunohistochemistry low-magnification photo of the liver mass in Case 3 (4×).
图12、病例3肝包块双染免疫组化中高倍照片（20× ）。Figure 12. Double-stained immunohistochemistry photo of the liver mass in Case 3 (20×).
具体实施方式Detailed ways
对本领域而言，在血液系统疾病的诊断中，可以通过新鲜标本的流式细胞术技术，检测某群细胞是否有相关蛋白标记的共表达或某些标记的缺失。但在实体肿瘤中，首先新鲜标本获取不易，其次也不是所有的免疫组化蛋白抗体标记均有流式细胞术抗体，故精准的诊断还是要依赖组织切片。因此如能在一张组织切片上同时辨认出胆管上皮的良性及恶性成分，对本领域而言有重大的实践意义，对一线临床的工作有很大的帮助。For this field, in the diagnosis of hematological diseases, flow cytometry technology on fresh specimens can be used to detect whether a certain group of cells has co-expression of related protein markers or the absence of certain markers. However, in solid tumors, first of all, it is difficult to obtain fresh specimens, and secondly, not all immunohistochemical protein antibody markers have flow cytometry antibodies, so accurate diagnosis still relies on tissue sections. Therefore, if the benign and malignant components of bile duct epithelium can be identified simultaneously on a tissue section, it will be of great practical significance to this field and will be of great help to front-line clinical work.
而本发明创造性地开发了针对两种不同的标志物EZH2和CK7双重免疫组化染色技术，发现选择EZH2和CK7作为抗原蛋白指标，进行不同颜色的抗原免疫染色，可在一张切片上同时辨认出胆管上皮的良性及恶性成分，加深对ICC及反应性胆管的进一步认识，有助于肿瘤的正确诊断。The present invention creatively developed a double immunohistochemical staining technology for two different markers, EZH2 and CK7, and found that selecting EZH2 and CK7 as antigenic protein indicators and performing antigen immunostaining of different colors can be identified simultaneously on one slice. Identify the benign and malignant components of bile duct epithelium, deepen our understanding of ICC and reactive bile ducts, and contribute to the correct diagnosis of tumors.
本发明双重免疫组化染色试剂盒。该试剂盒包括一抗试剂、二抗试剂 和染色剂；所述一抗试剂包括有EZH2抗体和CK7抗体；所述二抗试剂包括组合二抗（HRP标记抗小鼠IgG/HRP+兔抗IgG/AP，此二抗混合液可以让鼠来源一抗标记的部位与DAB显色为棕色而兔来源一抗标记的部位与AP红显色为红色）或组合二抗（HRP标记抗小鼠IgG/AP+兔抗IgG/HRP，此二抗混合液可以让鼠来源一抗标记的部位与AP红显色为红色而兔来源一抗标记的部位与DAB显色为棕色）。一般来说，所述EZH2抗体和CK7抗体最好来源于两种不同种属的动物。比如，两种不同种属的动物分别为鼠或兔。染色剂可以选用本领域常用的各种染色剂。比如使用广泛运用的过氧化物酶HRP 辣根介导的染色剂和碱性磷酸酶（AP）介导的染色剂。而对具体染色的操作进行一定的优化以获得更为对比更为清晰鲜明的成片也是需要进行的工作。Double immunohistochemical staining kit of the present invention. The kit includes primary antibody reagents, secondary antibody reagents and staining agents; the primary antibody reagents include EZH2 antibodies and CK7 antibodies; the secondary antibody reagents include combined secondary antibodies (HRP labeled anti-mouse IgG/HRP+rabbit anti-IgG/ AP, this secondary antibody mixture can make the part labeled with the mouse-derived primary antibody and DAB appear brown and the part labeled with the rabbit-derived primary antibody and AP red appear red) or a combination of secondary antibodies (HRP-labeled anti-mouse IgG/ AP+rabbit anti-IgG/HRP, this secondary antibody mixture can make the part labeled with the mouse primary antibody and AP red appear red and the part labeled with the rabbit primary antibody and DAB appear brown). Generally speaking, the EZH2 antibody and CK7 antibody are preferably derived from two different species of animals. For example, two different species of animals are rats or rabbits. The dyeing agent can be selected from various dyeing agents commonly used in this field. For example, the widely used peroxidase HRP horseradish-mediated stains and alkaline phosphatase (AP)-mediated stains are used. It is also necessary to optimize the specific dyeing operations to obtain a clearer and more contrasting film.
本发明方法能在同一张组织切片上分别或同时进行双重显色，染色对比鲜明直观。经过多因素（不同技术人员、不同组织等）对比验证，抗原没有丢失，抗体之间互不干扰，专家判读结果完全一致。具有很好的推广价值。The method of the invention can perform dual color development on the same tissue section separately or simultaneously, and the staining contrast is clear and intuitive. After comparison and verification by multiple factors (different technicians, different organizations, etc.), the antigen was not lost, the antibodies did not interfere with each other, and the expert interpretation results were completely consistent. It has very good promotion value.
以下通过的实施例对本发明技术方案进行进一步的详细说明，并非对本发明保护范围的限制。The following examples further illustrate the technical solutions of the present invention in detail and are not intended to limit the scope of the present invention.
本发明实施例中具体使用的主要试剂名称及其购买的厂家为：The names of the main reagents specifically used in the examples of the present invention and the manufacturers from which they were purchased are:
CK7一抗，克隆号：UMAB161，鼠单抗，即用型，中杉金桥；CK7 primary antibody, clone number: UMAB161, mouse monoclonal antibody, ready-to-use, Zhongshan Jinqiao;
EZH2一抗，克隆号：SP129，兔单抗，即用型，基因科技；EZH2 primary antibody, clone number: SP129, rabbit monoclonal antibody, ready to use, Gene Technology;
ultraView Universal DAB Detection Kit，Roche（罗氏），REF:760-500，其为Ventana染色平台使用的HRP标记的二抗试剂盒，试剂盒包括DAB显色液。ultraView Universal DAB Detection Kit, Roche (Roche), REF:760-500, is an HRP-labeled secondary antibody kit used on the Ventana staining platform. The kit includes DAB chromogenic solution.
ultraView Universal Alkaline Phosphatase Red Detection Kit，Roche（罗氏），REF:760-501，其为Ventana染色平台使用的AP标记二抗试剂盒，试剂盒包括AP红显色液。ultraView Universal Alkaline Phosphatase Red Detection Kit, Roche (Roche), REF:760-501, is an AP-labeled secondary antibody kit used on the Ventana staining platform. The kit includes AP red chromogenic solution.
Roche（罗氏）Ventana BenchMark 免疫组化仪。Roche (Roche) Ventana BenchMark immunohistochemistry instrument.
实施例1 使用本发明技术制备术后双染免疫组化切片Example 1 Preparation of postoperative double-stained immunohistochemical sections using the technology of the present invention
本实施例中的待诊断病例1为一名66岁女性患者，因“皮肤及巩膜黄染3+月，检查发现肝占位1+月”入院；术后病理参见图1和图2。Case 1 to be diagnosed in this example is a 66-year-old female patient who was admitted to the hospital due to "jaundice of the skin and sclera for3+ months, and liver occupation for1+ months"; the postoperative pathology is shown in Figures 1 and 2.
图1为HE低倍（4×）：肝被膜下见较多增生的胆管上皮，排列呈较不规则的实性条索状、鹿角或分枝状，间质疏松，纤维化不明显，边界不清楚。这些管腔到底是反应性的还是肿瘤性的，鉴别诊断包括：胆管腺瘤，肝内胆管癌（细胆管癌）。Figure 1 is HE low magnification (4×): there is a lot of proliferated bile duct epithelium under the liver capsule, arranged in more irregular solid cord-like, antler or branch-like shapes, with loose interstitium, inconspicuous fibrosis, and border Not sure. Whether these lumens are reactive or neoplastic, the differential diagnosis includes: bile duct adenoma, intrahepatic cholangiocarcinoma (cholangiocarcinoma).
图2为HE中倍（10×）：胆管细胞呈小立方状，类似细胆管上皮细胞，大小一致，部分胞质少，核浆比增大，核卵圆形，核分裂罕见，诊断困难。Figure 2 shows HE at medium magnification (10×): the bile duct cells are small cuboidal in shape, similar to thin bile duct epithelial cells, uniform in size, partially with less cytoplasm, increased nuclear-cytoplasmic ratio, oval nuclei, and rare mitoses, making diagnosis difficult.
为了能够进行清晰的诊断，对患者的组织进行了CK7和EZH2 双染免疫组化。In order to be able to make a clear diagnosis, CK7 and EZH2 double-stained immunohistochemistry was performed on the patient's tissue.
具体的制备的步骤如下：The specific preparation steps are as follows:
1、制备4μm的石蜡切片，65℃烤片30分钟；1. Prepare 4 μm paraffin sections and bake them at 65°C for 30 minutes;
2、使用Roche Ventana BenchMark 进行脱蜡和抗原修复；2. Use Roche Ventana BenchMark for dewaxing and antigen retrieval;
3、EZH2一抗滴加，孵育36分钟后ultraView Universal DAB Detection Kit二抗孵育并完成DAB显色；3. Add EZH2 primary antibody dropwise, and after incubation for 36 minutes, incubate with ultraView Universal DAB Detection Kit secondary antibody and complete DAB color development;
4、CK7一抗滴加，孵育64分钟后ultraView Universal Alkaline PhosphataseRed Detection Kit二抗孵育并完成AP红显色；4. Add CK7 primary antibody dropwise, and after incubation for 64 minutes, ultraView Universal Alkaline PhosphataseRed Detection Kit secondary antibody is incubated and AP red color development is completed;
5、苏木素复染4分钟后反蓝液反蓝；5. The anti-blue solution will turn blue after 4 minutes of hematoxylin counterstaining;
6、自来水冲洗后65℃烤箱烘干3小时，中性树胶封片后镜下观察。6. After rinsing with tap water, dry it in an oven at 65°C for 3 hours, seal it with neutral gum and observe it under a microscope.
2~5染色步骤均在Roche Ventana BenchMark染色平台操作完成。Staining steps 2~5 are all completed on the Roche Ventana BenchMark staining platform.
双染后的结果参见图3和图4。The results after double staining are shown in Figures 3 and 4.
图3为双染免疫组化低倍（4×）：可见周边胆管上皮及右侧胆管上皮CK7+EZH2-（蓝色箭头所指，显示红色，仅细胞膜染成红色），而中央及左上角则为红色+棕色，即CK7+EZH2+（红棕色，红色的细胞膜+棕色的细胞核），提示中央及左上角的胆管为胆管腺癌。而周边及右上角的则为反应性/残留的胆管上皮，而非肿瘤性的。Figure 3 shows double-stained immunohistochemistry at low magnification (4×): peripheral bile duct epithelium and right bile duct epithelium CK7+EZH2- can be seen (pointed by the blue arrow, showing red, only the cell membrane is stained red), while the center and upper left corner It is red + brown, that is, CK7+EZH2+ (red-brown, red cell membrane + brown cell nucleus), indicating that the bile ducts in the center and upper left corner are bile duct adenocarcinoma. The periphery and upper right corner are reactive/residual bile duct epithelium, not neoplastic.
图4为双染免疫组化中倍（10×）：蓝色箭头为反应性胆管上皮，红色箭头为胆管癌，对比分明，有助于诊断。Figure 4 shows double-stained immunohistochemistry at medium magnification (10×): the blue arrow indicates reactive bile duct epithelium and the red arrow indicates cholangiocarcinoma. The contrast is clear and helpful for diagnosis.
从该病例的各种染色图中可以看出，使用双染染色后的细胞形态清晰，细胞核、细胞膜和胞浆的颜色分明，阳性定位准确，信号表达清晰，能够应用于在胆管腺癌和反应性/残留的胆管上皮的鉴别诊断中。It can be seen from the various staining pictures of this case that the cell morphology after double staining is clear, the nuclei, cell membranes and cytoplasm are clearly colored, the positive positioning is accurate, and the signal expression is clear, which can be applied to bile duct adenocarcinoma and reactions. In the differential diagnosis of sexual/residual bile duct epithelium.
实施例2 使用本发明技术制备术后双染免疫组化切片Example 2 Preparation of postoperative double-stained immunohistochemical sections using the technology of the present invention
本实施例中的待诊断病例2为一名60岁男性患者，因“体检发现肝占位20天”入院；Case 2 to be diagnosed in this example is a 60-year-old male patient who was admitted to the hospital because "a physical examination revealed that the liver has occupied space for 20 days";
术后病理参见图5至图7。The postoperative pathology is shown in Figures 5 to 7.
图5为HE中倍（10×）：可见梭形细胞排列呈束状及片状，异型性明显。其内隐约可见散在的单层立方上皮形成的管腔样结构或条索样结构，细胞有一定的异型性。这些隐约可见的管腔到底是反应性的还是肿瘤性的，不同的医生各执一词、争议较大。诊断考虑为梭形细胞恶性肿瘤。鉴别诊断包括：肉瘤样肝细胞癌、肝内胆管癌+肉瘤样癌、肉瘤等。Figure 5 is HE medium magnification (10×): it can be seen that the spindle cells are arranged in bundles and sheets, with obvious atypia. There are faintly visible lumen-like structures or cord-like structures formed by scattered single-layer cuboidal epithelium, and the cells have certain atypia. Whether these faintly visible lumens are reactive or neoplastic is a matter of dispute among different doctors. The diagnosis was considered spindle cell malignancy. Differential diagnosis includes: sarcomatoid hepatocellular carcinoma, intrahepatic cholangiocarcinoma + sarcomatoid carcinoma, sarcoma, etc.
图6为单染的CK7免疫组化中倍（10×）：CK7示瘤体中胆管上皮，可见腺样排列、少数不规则，可见鹿角样及不规则型，但仍不清楚这些胆管到底是反应性的还是肿瘤性的。Figure 6 is a single-stained CK7 immunohistochemistry medium magnification (10×): CK7 shows bile duct epithelium in the tumor, with glandular arrangement, a few irregularities, antler-like and irregular patterns, but it is still unclear what these bile ducts are. Reactive or neoplastic.
图7为单染的EZH2免疫组化中倍（10×）：EZH2示瘤体中梭形细胞高表达，经反复对比HE切片，CK7的免疫组化，找到大致相同部位的胆管上皮，经仔细对比辨认，可见胆管上皮细胞为阴性表达（箭头所指）。Figure 7 shows the single-stained EZH2 immunohistochemistry medium magnification (10×): EZH2 shows high expression of spindle cells in the tumor. After repeated comparison of HE sections and CK7 immunohistochemistry, bile duct epithelium was found in roughly the same location. After careful Comparative identification shows that the bile duct epithelial cells have negative expression (arrowed).
为了能够进行清晰的诊断，是用本发明技术对组织进行了CK7和EZH2 双染免疫组化。In order to make a clear diagnosis, CK7 and EZH2 double-stained immunohistochemistry was performed on the tissue using the technology of the present invention.
具体的染色成片的步骤如下：The specific steps for dyeing into pieces are as follows:
1、制备4μm的石蜡切片，65℃烤片30分钟；1. Prepare 4 μm paraffin sections and bake them at 65°C for 30 minutes;
2、使用Roche Ventana BenchMark 进行脱蜡和抗原修复；2. Use Roche Ventana BenchMark for dewaxing and antigen retrieval;
3、EZH2一抗滴加，孵育36分钟后ultraView Universal DAB Detection Kit二抗孵育并完成DAB显色；3. Add EZH2 primary antibody dropwise, and after incubation for 36 minutes, incubate with ultraView Universal DAB Detection Kit secondary antibody and complete DAB color development;
4、CK7一抗滴加，孵育64分钟后ultraView Universal Alkaline PhosphataseRed Detection Kit二抗孵育并完成AP红显色；4. Add CK7 primary antibody dropwise, and after incubation for 64 minutes, ultraView Universal Alkaline PhosphataseRed Detection Kit secondary antibody is incubated and AP red color development is completed;
5、苏木素复染4分钟后反蓝液反蓝；5. The anti-blue solution will turn blue after 4 minutes of hematoxylin counterstaining;
6、自来水冲洗后65℃烤箱烘干3小时，中性树胶封片后镜下观察。6. After rinsing with tap water, dry it in an oven at 65°C for 3 hours, seal it with neutral gum and observe it under a microscope.
2~5染色步骤均在Roche Ventana BenchMark染色平台操作完成。Staining steps 2~5 are all completed on the Roche Ventana BenchMark staining platform.
双染后的结果参见图8。图8为双重染色免疫组化中高倍照片（20×），可见单层立方上皮围绕形成的管腔样结构CK7+EZH2-（红色，细胞膜）、和梭形细胞CK7-EZH2+（棕色，细胞核）在一张切片上显示，颜色对比鲜明，不需要反复对比查看。这表明梭形细胞肿瘤中存在的胆管上皮为反应性/或残留的胆管上皮，而不是恶性成分。即不是ICC，也不是混合性HCC-ICC，有助于正确的病理诊断。而且可见使用双染染色后的细胞形态清晰，细胞核、细胞膜和细胞浆的颜色分明，阳性定位准确，信号表达清晰，能够应用于在胆管腺癌和反应性/残留的胆管上皮的鉴别诊断中。The results after double staining are shown in Figure 8. Figure 8 is a double-stained immunohistochemistry medium-high magnification photo (20×), showing the lumen-like structure CK7+EZH2- (red, cell membrane) formed by a single layer of cuboidal epithelium, and the spindle cells CK7-EZH2+ (brown, cell nucleus). Displayed on one slice, the colors are contrasting and do not require repeated comparison and viewing. This suggests that the bile duct epithelium present in spindle cell tumors is reactive/or residual bile duct epithelium rather than a malignant component. That is, it is not ICC, nor is it mixed HCC-ICC, which is helpful for correct pathological diagnosis. Moreover, it can be seen that the cell morphology after double staining is clear, the nuclei, cell membranes and cytoplasm are clearly colored, the positive positioning is accurate, and the signal expression is clear. It can be applied in the differential diagnosis of bile duct adenocarcinoma and reactive/residual bile duct epithelium.
实施例3 使用本发明技术制备术后双染免疫组化切片Example 3 Using the technology of the present invention to prepare postoperative double-stained immunohistochemical sections
本实施例中的待诊断病例3为一名39岁女性患者，因“间断腹胀腹痛10+年，确诊胃窦腺癌1月”入院手术；术中发现左肝缘表面见1枚直径约0.3cm灰白色结节，术中取该结节送病理，希望明确是否为胃原发的转移性腺癌。术后石蜡切片病理参见图9和图10。Case 3 to be diagnosed in this example is a 39-year-old female patient who was admitted to the hospital for surgery due to "intermittent abdominal distension and abdominal pain for10+ years, and was diagnosed with gastric antral adenocarcinoma for 1 month"; during the operation, it was found that a diameter of about 0.3 was found on the surface of the left liver edge. cm gray-white nodule, the nodule was removed during surgery and sent to pathology, hoping to determine whether it was primary metastatic adenocarcinoma of the stomach. The postoperative paraffin section pathology is shown in Figures 9 and 10.
图9为HE低倍（4×）：肝被膜下见孤立结节，为较多增生的胆管上皮，大小较一致，管腔狭小，弯曲状或呈较不规则的实性条索状，无囊性空腔或胆管扩张，间质可见淋巴细胞浸润，边界欠清楚。这些管腔到底是反应性的还是肿瘤性的，鉴别诊断包括：胆管腺瘤，肝内胆管癌（细胆管癌），转移性腺癌。Figure 9 shows HE low magnification (4×): Solitary nodules are seen under the liver capsule, which are more proliferated bile duct epithelial cells with uniform size, narrow lumen, curved shape or more irregular solid strip shape, no Cystic cavities or bile ducts are dilated, lymphocyte infiltration can be seen in the interstitium, and the boundaries are unclear. Whether these lumens are reactive or neoplastic, the differential diagnosis includes: bile duct adenoma, intrahepatic cholangiocarcinoma (cholangiocarcinoma), and metastatic adenocarcinoma.
图10为HE中倍（10×）：可见胆管细胞呈立方状或矮柱状，无明显异型性，胞浆丰富，细胞核小圆形，大小均匀，无明显核分裂像，诊断困难。Figure 10 is HE medium magnification (10×): it can be seen that the bile duct cells are cubic or short columnar, with no obvious atypia, abundant cytoplasm, small round nuclei, uniform size, and no obvious mitotic figures, making diagnosis difficult.
为了能够进行清晰的诊断，对患者组织进行了CK7和EZH2 双染免疫组化。本实例中仍按以上实施例的染色方案完成双染免疫组化染色。双染后的结果参见图11和图12。To enable a clear diagnosis, CK7 and EZH2 double-stained immunohistochemistry was performed on patient tissues. In this example, the double-stained immunohistochemical staining is still completed according to the staining scheme of the above example. The results after double staining are shown in Figure 11 and Figure 12.
图11为双重染色免疫组化低倍照片（10×），可见单层立方上皮围绕形成的管腔样结构CK7+EZH2-（红色，细胞膜）、并显示周边肝组织的门管区的正常胆管上皮，而未见CK7+EZH2+（棕色，细胞核）的棕色胆管上皮，颜色鲜明。表明这些胆管上皮为良性病变，而不是恶性成分。有助于正确的病理诊断。Figure 11 is a low-magnification photo of double staining immunohistochemistry (10×), showing the lumen-like structure CK7+EZH2- (red, cell membrane) formed by a single layer of cuboidal epithelium, and the normal bile duct epithelium in the portal area of the surrounding liver tissue. , while the brown bile duct epithelium with CK7+EZH2+ (brown, cell nuclei) is not seen, and the color is bright. This indicates that these bile duct epithelia are benign lesions rather than malignant components. Contribute to correct pathological diagnosis.
图12为双重染色免疫组化中高倍照片（20×），可见单层立方上皮围绕形成的管腔样结构CK7+EZH2-（红色，细胞膜）、CK7-EZH2+（棕色，细胞核）为反应性的淋巴细胞，也可以当成自身阳性对照，二者在一张切片上显示，颜色对比鲜明，可见使用双染染色后的细胞形态清晰，细胞核、细胞膜和细胞浆的颜色分明，阳性定位准确，信号表达清晰，能够应用于在胆管腺癌、反应性/残留的胆管上皮及转移性腺癌的鉴别诊断中。Figure 12 is a double-stained immunohistochemistry medium-high magnification photo (20×). It can be seen that the lumen-like structure formed by a single layer of cuboidal epithelium CK7+EZH2- (red, cell membrane) and CK7-EZH2+ (brown, cell nucleus) are reactive. Lymphocytes can also be used as their own positive controls. The two are displayed on one section with sharp color contrast. It can be seen that the cell shape after double staining is clear, the color of the nucleus, cell membrane and cytoplasm is clear, the positive positioning is accurate, and the signal expression is It is clear and can be used in the differential diagnosis of bile duct adenocarcinoma, reactive/residual bile duct epithelium and metastatic adenocarcinoma.
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