人肺组织中ZW10相互作用蛋白免疫染色的数字化分析
1Department of Hematology,Zhongnan Hospital of Wuhan University, 2Department of Thoracic Surgery,Renmin Hospital of Wuhan University, 3Department of Human Anatomy, Histology and Embryology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine,Peking Union Medical College, 4Department of Immunology, School of Basic Medical Sciences,Wuhan University, 5Department of Gastroenterology,Central Hospital of Wuhan, 6Department of Transfusion,Zhongnan Hospital of Wuhan University
Summary
ZW10相互作用蛋白(ZWINT)参与线粒主轴检查点和癌的发病机制。在这里,我们介绍了一种在人类肺癌组织中对ZWINT进行免疫染色的方法,然后对整个幻灯片进行数字扫描和图像分析。该方法可以提供高质量的数字图像和可靠的结果。
Abstract
本研究旨在介绍人体肺组织的免疫染色方法,然后进行全滑数字扫描和图像分析。数字扫描是扫描一叠幻灯片并生成高质量数字图像的快速方法。它可以产生与病理学家的传统光显微镜(CLM)一致的结果。此外,数字图像的可用性使得多人可以同时观察到同一张幻灯片。此外,幻灯片的数字图像可以存储在数据库中,这意味着玻璃幻灯片的长期恶化是可以避免的。此技术的局限性如下。首先,它需要高质量的制备组织和原始的免疫组织化学(IHC)幻灯片,没有任何损伤或过多的密封剂残留物。其次,肿瘤或非肿瘤区域应在使用软件进行分析之前由经验丰富的病理学家指定,以避免在评分过程中对肿瘤或非肿瘤区域产生任何混淆。第三,操作员需要在整个幻灯片成像过程中控制色彩再现。
Introduction
ZW10相互作用蛋白(ZWINT)是血吸管复合物的必要成分,它涉及线粒主轴检查点1,2,3。据报道,ZWINT的耗竭导致异常的过早染色体分离1,2,3。最近的研究表明,ZWINT通过促进肿瘤细胞4、5的增殖参与多种肿瘤的发病机制。我们之前曾报道过,ZWINT在肺癌5中过度表达。人们普遍认为,病理学家使用CLM分析幻灯片是费时费时的,而不是定量的6,7,8。 此外,存储的玻璃幻灯片的损坏可能使其无法收回以前创建的幻灯片。基于计算机的数字化全幻灯片成像(WSI)的新兴方法可以克服这些限制6,7,8。
为此,我们描述了人类肺癌组织中ZWINT免疫染色的方法,以及全幻灯片数字扫描和基于软件的图像分析。该方法的主要优点是与 CLM 产生一致的结果。该技术可广泛应用于血氧素-eosin染色(H&E)和IHC的病理评分、原位杂交(FISH)、组织微阵列(TMA)以及药物发现和开发等领域。
Protocol
本文介绍的所有方法均已经武汉大学中南医院和武汉大学人民医院伦理委员会批准。 1. 准备 IHC 幻灯片 将人体肺组织片段(约3 x 3 cm)浸入磷酸盐缓冲盐水(PBS)中的4%甲醛中,在室温(RT)下24小时,修复肺组织样本。 在室温下,分别在80%、95%和100%乙醇中脱水组织15分钟、20分钟和20分钟。最后,在室温下将组织浸入100%二甲苯3倍,每次20分钟。 对于嵌入,将组织浸入石?…
Representative Results
通过IHC,我们测量了28对非小细胞肺癌(NSCLC)标本(肿瘤和相邻非肿瘤组织)的ZWINT表达水平,包括14个鳞状细胞癌(SCC)和14个腺癌(ADC)。幻灯片全滑动数字扫描提供了高质量的数字图像(图1A)。结果表明,肺癌的H-评分明显高于相邻非癌组织(P<0.0001,双尾t-test)(图1A和1B)。此外,我们发现CCC中的ZWINT表达水平明显高于ADC…
Discussion
全幻灯片扫描正成为一个热门话题,其强大的扫描和生产高品质的图像,用于临床和研究目的11,12,13。图像可以在11、12、13分钟之内通过滑动扫描显微镜产生。通过应用该方法,我们获得了ZWINT IHC幻灯片的高质量图像,并比较了肿瘤组织和非肿瘤组织的H-分数。在…
Declarações
The authors have nothing to disclose.
Acknowledgements
该项目得到了中国国家自然基金会(第81500151号、81400121号、81270607、81541027和81501352)和湖北省自然基金会(中国)(第2017CFB631号)的支持。作者对郭勤、常敏、李辉及其武汉谷歌生物科技有限公司同事的技术支持表示感谢。作者还感谢穆罕默德·贾迈勒的语言编辑。
Materials
| Pannoramic MIDI | 3D HISTECH | Cat: PMIDI-040709 | An automated digital slide scanner with a remarkable feature set :12-slide capacity, fluorescence scanning, and many more. |
| QuantCenter | 3D HISTECH | Downloaded from the official website of the company | The framework for 3DHISTCH's image analysis applications. |
| LEICA RM2235 | Leica Microsystems | Cat: 14050038604 | The enhanced precision of the new accessories will add convenience to block to knife approach as well as specimen orientation. |
| Rabbit anti-human Anti-ZWINT antibody | Abcam | Cat: ab197794 | Immunohistochemical analysis of ZWINT in human lung tissue. |
| Anti-rabbit secondary antibody | Wuhan Goodbio Technology | Cat:GB23303-1 | Secondary antibody for IHC staining. |
| Phosphate-buffered saline | Wuhan Goodbio Technology | Cat:G0002 | A solution containing a phosphate buffer. |
| OLYMPUS CX23 | OLYMPUS | Cat:6M87620 | Microscope for detection of H&E or IHC slides. |
| Dimethylbenzene | Shanghai Lingfeng Chemical Reagent | Cat:1330-20-7 | A colorless, flammable fluid used as a solvent and clarifying agent in the preparation of tissue sections for microscopic study. |
| Hematoxylin Staining Solution | Wuhan Servicebio technology | Cat:G1039 | It is commonly used for histologic studies, oftern colors the nuclei of cells blue. |
| Tween 20 | Baitg | Cat:2005-64-5 | It is a polysorbate-type nonionic surfactant formed by the ethoxylation of sorbitan before the addition of lauric acid. It is used as a deterent and emulsifier in pharmacological applications. |
| Citric acid repair liquid | Wuhan Servicebio technology | Cat:G1202 | Is is used to repair antigen after fixation during IHC procedure. |
| LEICA ASP200s | Leica | Cat: 14048043626 | It was designed for routine and research histopathology of up to 200 cassettes. |
| LEICA Arcadia H | Leica | Cat: 14039354103 | It is a heated paraffin embedding station and allows for simple operation and precise control, resulting in improved quality, a smooth workflow and reliability. |
| LEICA Arcadia C | Leica | Cat: 14039354102 | It is a cold plate holding more than 60/65 cassettes on its large working surface. It was designed with an environment adaptive control module to make sure the operating temperature is always stabilized at -6°C. |
| CaseViewer Software | 3DHISTECH |
Referências
- Endo, H., Ikeda, K., Urano, T., Horie-Inoue, K., Inoue, S. Terf/TRIM17 stimulates degradation of kinetochore protein ZWINT and regulates cell proliferation. The Journal of Biochemistry. 151 (2), 139-144 (2012).
- Wang, H., et al. Human Zwint-1 specifies localization of Zeste White 10 to kinetochores and is essential for mitotic checkpoint signaling. Journal of Biological Chemistry. 279 (52), 54590-54598 (2004).
- Lin, Y. T., Chen, Y., Wu, G., Lee, W. H. Hec1 sequentially recruits Zwint-1 and ZW10 to kinetochores for faithful chromosome segregation and spindle checkpoint control. Oncogene. 25 (52), 6901-6914 (2006).
- Ying, H., et al. Overexpression of Zwint predicts poor prognosis and promotes the proliferation of hepatocellular carcinoma by regulating cell-cycle-related proteins. OncoTargets and Therapy. 11, 689-702 (2018).
- Yuan, W., et al. Bioinformatic analysis of prognostic value of ZW10 interacting protein in lung cancer. OncoTargets and Therapy. 11, 1683-1695 (2018).
- Higgins, C. Applications and challenges of digital pathology and whole slide imaging. Biotechnic & Histochemistry. 90 (5), 341-347 (2015).
- Webster, J. D., Dunstan, R. W. Whole-slide imaging and automated image analysis: considerations and opportunities in the practice of pathology. Veterinary Pathology. 51 (1), 211-223 (2014).
- Al-Janabi, S., Huisman, A., van Diest, P. J. Digital pathology: current status and future perspectives. Histopathology. 61 (1), 1-9 (2012).
- Bonomi, P. D., et al. Predictive biomarkers for response to EGFR-directed monoclonal antibodies for advanced squamous cell lung cancer. Annals of Oncology. 29 (8), 1701-1709 (2018).
- Villalobos, M., et al. ERCC1 assessment in upfront treatment with and without cisplatin-based chemotherapy in stage IIIB/IV non-squamous non-small cell. Medical Oncology. 35 (7), 106 (2018).
- Griffin, J., Treanor, D. Digital pathology in clinical use: where are we now and what is holding us back?. Histopathology. 70 (1), 134-145 (2017).
- Huisman, A., Looijen, A., van den Brink, S. M., van Diest, P. J. Creation of a fully digital pathology slide archive by high-volume tissue slide scanning. Human Pathology. 41 (5), 751-775 (2010).
- Gray, A., Wright, A., Jackson, P., Hale, M., Treanor, D. Quantification of histochemical stains using whole slide imaging: development of a method and demonstration of its usefulness in laboratory quality control. Journal of Clinical Pathology. 68 (3), 192-199 (2015).
- Hofman, F. M., Taylor, C. R. Immunohistochemistry. Current Protocols in Immunology. 103, (2013).
- Ramos-Vara, J. A. Principles and Methods of Immunohistochemistry. Methods in Molecular Biology. 1641, 115-128 (2017).
- Otali, D., Fredenburgh, J., Oelschlager, D. K., Grizzle, W. E. A standard tissue as a control for histochemical and immunohistochemical staining. Biotechnic & Histochemistry. 91 (5), 309-326 (2016).
- Clarke, E. L., Treanor, D. Colour in digital pathology: a review. Histopathology. 70 (2), 153-163 (2017).
- Potts, S. J. Digital pathology in drug discovery and development: multisite integration. Drug Discovery Today. 14 (19-20), 935-941 (2009).
- Tabata, K., et al. Whole-slide imaging at primary pathological diagnosis: Validation of whole-slide imaging-based primary pathological diagnosis at twelve Japanese academic institutes. Pathology International. 67 (11), 547-554 (2017).
- Saco, A., Bombi, J. A., Garcia, A., Ramírez, J., Ordi, J. Current Status of Whole-Slide Imaging in Education. Pathobiology. 83 (2-3), 79-88 (2016).
- Griffin, J., Treanor, D. Digital pathology in clinical use: where are we now and what is holding us back?. Histopathology. 70 (1), 134-145 (2017).
Citar este artigo
Wen, Y., Song-ping, X., Pan, L., Xiao-yan, L., Shan, P., Qian, Y., Meng, S., Xiao-xing, H., Rui-jing, X., Jie, X., Qiu-ping, Z., Liang, S. Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues. J. Vis. Exp. (147), e58551, doi:10.3791/58551 (2019).