Name: digital analysis of immunostaining of zw10 interacting protein in human lung tissues
Uploaded: 2019-05-01T13:00:00
Description: Watch this Scientific Journal Video about Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues at JoVE.com

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.

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
<ol>
	<li>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).</li>
	<li>Dehydrate the tissue in 80%, 95%, and 100% ethanol for 15 min, 20 min, and 20 min, respectively, at room temperat...

<ol>
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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...

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 overexpression of ZWINT in lung cancer5. It has been widely accepted that the analysis of slides by pathologists using CLM is time-consuming and not quantitative6,7,8. Moreover, the deterioration of stored glass slides might make it impossible to retract previously created slides. The emerging method of computer-based, digital whole-slide imaging (WSI) may overcome these limitations6,7,8.
To this end, we describe a methodology of the immunostaining of ZWINT in human lung cancer tissues, coupled with whole-slide digital scanning and software-based image analysis. The main advantage of this methodology is the production of concordant results with CLM. This technology can be widely used in the areas of pathological scoring of hematoxylin-eosin staining (H&#38;E) and IHC, fluorescence in situ hybridization (FISH), tissue microarrays (TMA), and drug discovery and development.

<table>
	<tbody>
		<tr>
			<td>Pannoramic MIDI</td>
			<td>3D HISTECH</td>
			<td>Cat: PMIDI-040709</td>
			<td>An automated digital slide scanner with a remarkable feature set :12-slide capacity, fluorescence scanning, and many more.</td>
		</tr>
		<tr>
			<td>QuantCenter</td>
			<td>3D HISTECH</td>
			<td>Downloaded from the official website of the company</td>
			<td>The framework for 3DHISTCH&#39;s image analysis applications.</td>
		</tr>
		<tr>
			<td>LEICA RM2235</td>
			<td>Leica Microsystems</td>
			<td>Cat: 14050038604</td>
			<td>The enhanced precision of the new accessories will add convenience to block to knife approach as well as specimen orientation.</td>
		</tr>
		<tr>
			<td>Rabbit anti-human Anti-ZWINT antibody</td>
			<td>Abcam</td>
			<td>Cat: ab197794</td>
			<td>Immunohistochemical analysis of ZWINT in human lung tissue.</td>
		</tr>
		<tr>
			<td>Anti-rabbit secondary antibody</td>
			<td>Wuhan Goodbio Technology</td>
			<td>Cat:GB23303-1</td>
			<td>Secondary antibody for IHC staining.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Wuhan Goodbio Technology</td>
			<td>Cat:G0002</td>
			<td>A solution containing a phosphate buffer.</td>
		</tr>
		<tr>
			<td>OLYMPUS CX23</td>
			<td>OLYMPUS</td>
			<td>Cat:6M87620</td>
			<td>Microscope for detection of H&#38;E or IHC slides.</td>
		</tr>
		<tr>
			<td>Dimethylbenzene</td>
			<td>Shanghai Lingfeng Chemical Reagent</td>
			<td>Cat:1330-20-7</td>
			<td>A colorless, flammable fluid used as a solvent and clarifying agent in the preparation of tissue sections for microscopic study.</td>
		</tr>
		<tr>
			<td>Hematoxylin Staining Solution</td>
			<td>Wuhan Servicebio technology</td>
			<td>Cat:G1039</td>
			<td>It is commonly used for histologic studies, oftern colors the nuclei of cells blue.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Baitg</td>
			<td>Cat:2005-64-5</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Citric acid repair liquid</td>
			<td>Wuhan Servicebio technology</td>
			<td>Cat:G1202</td>
			<td>Is is used to repair antigen after fixation during IHC procedure.</td>
		</tr>
		<tr>
			<td>LEICA ASP200s</td>
			<td>Leica</td>
			<td>Cat: 14048043626</td>
			<td>It was designed for routine and research histopathology of up to 200 cassettes.</td>
		</tr>
		<tr>
			<td>LEICA Arcadia H</td>
			<td>Leica</td>
			<td>Cat: 14039354103</td>
			<td>It is a heated paraffin embedding station and allows for simple operation and precise control, resulting in improved quality, a smooth workflow and reliability.</td>
		</tr>
		<tr>
			<td>LEICA Arcadia C</td>
			<td>Leica</td>
			<td>Cat: 14039354102</td>
			<td>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&#176;C.</td>
		</tr>
		<tr>
			<td>CaseViewer Software</td>
			<td>3DHISTECH</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

digital analysis of immunostaining of zw10 interacting protein in human lung tissues

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.

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 &#60; ...

Watch this Scientific Journal Video about Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues at JoVE.com

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

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 ...

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