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Cancer Research

Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues

Published: May 1st, 2019

DOI:

10.3791/58551

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

ZW10 interacting protein (ZWINT) participates in the mitotic spindle checkpoint and the pathogenesis of carcinoma. Here, we introduce a methodology of the immunostaining of ZWINT in human lung cancer tissues, followed by the digital scanning of whole slides and image analysis. This methodology can provide high-quality digital images and reliable results.

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image analysis. Digital scanning is a fast way to scan a stack of slides and produce digital images with high quality. It can produce concordant results with conventional light microscopy (CLM) by pathologists. Furthermore, the availability of digital images makes it possible that the same slide can be concurrently observed by multiple people. Moreover, digital images of slides can be stored in a database, which means the long-term deterioration of glass slides is avoided. The limitations of this technique are as follows. First, it needs high-quality prepared tissue and the original immunohistochemistry (IHC) slides without any damage or excess sealant residue. Second, tumor or nontumor areas should be specified by experienced pathologists before the analysis using software, in order to avoid any confusion about the tumor or nontumor areas during scoring. Third, the operator needs to control the color reproduction throughout the digitization process in whole-slide imaging.

ZW10 interacting protein (ZWINT) is a necessary component of the kinetochore complex which is involved in the mitotic spindle checkpoint1,2,3. It has been reported that the depletion of ZWINT leads to aberrant premature chromosome segregation1,2,3. Recent studies have suggested that ZWINT is involved in the pathogenesis of multiple tumors by promoting the proliferation of tumor cells4,5. We previously reported the overexpress....

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All methods described here have been approved by the Ethical Committee of Zhongnan Hospital of Wuhan University and Renmin Hospital of Wuhan University.

1. Preparation of IHC Slides

  1. Fix the lung tissue sample by immersing the human lung tissue fragment (about 3 x 3 cm) in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h at room temperature (RT).
  2. Dehydrate the tissue in 80%, 95%, and 100% ethanol for 15 min, 20 min, and 20 min, respectively, at room temperat.......

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We measured the expression levels of ZWINT in 28 pairs of non-small-cell lung cancer (NSCLC) specimens (tumor and adjacent nontumor tissues), including 14 squamous cell carcinomas(SCCs) and 14 adenocarcinomas (ADCs), by IHC. The whole-slide digital scanning of the slides provided digital images of high quality (Figure 1A). The results showed that the H-score of the lung cancer was significantly higher than that of adjacent noncancer tissues (P < .......

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Whole-slide scanning is becoming a hot topic for its robust scanning and production of high-quality images for clinical and research purposes11,12,13. Images can be produced by slide-scanning microscopes within minutes11,12,13. By applying this methodology, we obtained high-quality images for ZWINT IHC slides and compared the H-score be.......

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This project was supported by the National Natural Foundation of China (No. 81500151, 81400121, 81270607, 81541027, and 81501352) and the Natural Foundation of Hubei Province (China) (No. 2017CFB631). The authors express their appreciation to Guo Qin, Chang Min, Li Hui, and their colleagues at Wuhan Google Biological Technology Co., LTD for their technical support. The authors also thank Muhammad Jamal for the language editing.

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Name Company Catalog Number Comments
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

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