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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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hec1+sequentially+recruits+Zwint-1+and+ZW10+to+kinetochores+for+faithful+chromosome+segregation+and+spindle+checkpoint+control.">Hec1 sequentially recruits Zwint-1 and ZW10 to kinetochores for faithful chromosome segregation and spindle checkpoint control.</a> Oncogene. 25 (52), 6901-6914 (2006).</li><li>Ying, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Zwint+predicts+poor+prognosis+and+promotes+the+proliferation+of+hepatocellular+carcinoma+by+regulating+cell-cycle-related+proteins.">Overexpression of Zwint predicts poor prognosis and promotes the proliferation of hepatocellular carcinoma by regulating cell-cycle-related proteins.</a> OncoTargets and Therapy. 11, 689-702 (2018).</li><li>Yuan, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatic+analysis+of+prognostic+value+of+ZW10+interacting+protein+in+lung+cancer.">Bioinformatic analysis of prognostic value of ZW10 interacting protein in lung cancer.</a> OncoTargets and Therapy. 11, 1683-1695 (2018).</li><li>Higgins, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+challenges+of+digital+pathology+and+whole+slide+imaging.">Applications and challenges of digital pathology and whole slide imaging.</a> Biotechnic &amp; Histochemistry. 90 (5), 341-347 (2015).</li><li>Webster, J. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+biomarkers+for+response+to+EGFR-directed+monoclonal+antibodies+for+advanced+squamous+cell+lung+cancer.">Predictive biomarkers for response to EGFR-directed monoclonal antibodies for advanced squamous cell lung cancer.</a> Annals of Oncology. 29 (8), 1701-1709 (2018).</li><li>Villalobos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERCC1+assessment+in+upfront+treatment+with+and+without+cisplatin-based+chemotherapy+in+stage+IIIB/IV+non-squamous+non-small+cell.">ERCC1 assessment in upfront treatment with and without cisplatin-based chemotherapy in stage IIIB/IV non-squamous non-small cell.</a> Medical Oncology. 35 (7), 106 (2018).</li><li>Griffin, J., Treanor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+pathology+in+clinical+use:+where+are+we+now+and+what+is+holding+us+back?.">Digital pathology in clinical use: where are we now and what is holding us back?.</a> Histopathology. 70 (1), 134-145 (2017).</li><li>Huisman, A., Looijen, A., van den Brink, S. M., van Diest, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+fully+digital+pathology+slide+archive+by+high-volume+tissue+slide+scanning.">Creation of a fully digital pathology slide archive by high-volume tissue slide scanning.</a> Human Pathology. 41 (5), 751-775 (2010).</li><li>Gray, A., Wright, A., Jackson, P., Hale, M., Treanor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+histochemical+stains+using+whole+slide+imaging:+development+of+a+method+and+demonstration+of+its+usefulness+in+laboratory+quality+control.">Quantification of histochemical stains using whole slide imaging: development of a method and demonstration of its usefulness in laboratory quality control.</a> Journal of Clinical Pathology. 68 (3), 192-199 (2015).</li><li>Hofman, F. M., Taylor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry.">Immunohistochemistry.</a> Current Protocols in Immunology. 103, (2013).</li><li>Ramos-Vara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+Methods+of+Immunohistochemistry.">Principles and Methods of Immunohistochemistry.</a> Methods in Molecular Biology. 1641, 115-128 (2017).</li><li>Otali, D., Fredenburgh, J., Oelschlager, D. K., Grizzle, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standard+tissue+as+a+control+for+histochemical+and+immunohistochemical+staining.">A standard tissue as a control for histochemical and immunohistochemical staining.</a> Biotechnic &amp; Histochemistry. 91 (5), 309-326 (2016).</li><li>Clarke, E. L., Treanor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colour+in+digital+pathology:+a+review.">Colour in digital pathology: a review.</a> Histopathology. 70 (2), 153-163 (2017).</li><li>Potts, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+pathology+in+drug+discovery+and+development:+multisite+integration.">Digital pathology in drug discovery and development: multisite integration.</a> Drug Discovery Today. 14 (19-20), 935-941 (2009).</li><li>Tabata, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-slide+imaging+at+primary+pathological+diagnosis:+Validation+of+whole-slide+imaging-based+primary+pathological+diagnosis+at+twelve+Japanese+academic+institutes.">Whole-slide imaging at primary pathological diagnosis: Validation of whole-slide imaging-based primary pathological diagnosis at twelve Japanese academic institutes.</a> Pathology International. 67 (11), 547-554 (2017).</li><li>Saco, A., Bombi, J. A., Garcia, A., Ram&#237;rez, J., Ordi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Status+of+Whole-Slide+Imaging+in+Education.">Current Status of Whole-Slide Imaging in Education.</a> Pathobiology. 83 (2-3), 79-88 (2016).</li><li>Griffin, J., Treanor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+pathology+in+clinical+use:+where+are+we+now+and+what+is+holding+us+back?.">Digital pathology in clinical use: where are we now and what is holding us back?.</a> Histopathology. 70 (1), 134-145 (2017).</li></ol>

The authors have nothing to disclose.

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 ...

This method can help answer key questions in the pathological and the immunohistochemistry. The main advantage of this technique is that faster enough and that produces digital media with high quality. Begin by fixing a three-by-three centimeter kung tissue sample in 4%paraformaldehyde for 24 hours at room temperature.The next day, dehydrate the tissue in ascending ethanol immersions at room temperature, followed by three immersions in 100%xylene for 20 minutes per treatment. After the last xylene immersion, place the tissue in 63-degree Celsius paraffin for 30 minutes, followed by a second 45-minute immersion in fresh 63-degree Celsius paraffin, then change the paraffin one more time for a final one-hour incubation. When the paraffin has cooled, use a rotary microtome to acquire five micrometer-thick sections of the paraffin-embedded tissue sample, capturing the sections on 20 microgram-per-milliliter poly-L-lysine-coated slides.For dewaxing, place the slides in a 65-degree Celsius oven for two hours, followed by immersion in dimethylbenzene for 15 minutes and soaking in 100%xylene for 15 minutes, then rehydrating the slides in a descending ethanol immersion series. For antigen repair, place the slides in an antigen-repair box and add 300 milliliters of citric acid repair liquid to the box. Heat the box in a microwave oven at high power for two minutes, followed by heating at low power to sustain boiling for six minutes.When the slides have cooled to room temperature, wash the tissue samples with three five-minute 0.01 molar PBS washes in a rotary shaker at room temperature. To eliminate the endogenous peroxidase, incubate the slides with 3%hydrogen peroxide in double-distilled water for 30 minutes in a wet box at room temperature. At the end of the incubation, wash the slides as demonstrated and plug any nonspecific binding on each section with 100 microliters of 10%goat serum for 39 minutes at 37 degrees Celsius.Next, label the tissues with 100 microliters of anti-ZWINT antibody per slide at four degrees Celsius overnight, followed by three washes in 0.01 molar PBS. After the last wash, incubate the slides with an appropriate secondary antibody for 30 minutes at 37 degrees Celsius, followed by three 0.01 molar PBS washes. After the last wash, add the slides to 300 microliters of fresh DAB for 10 minutes, followed by a 10-second wash with tap water at room temperature.Counters stain the tissues with hematoxylin staining solution for two minutes at room temperature, followed by washing in tap water for 15 minutes, then dehydrate the tissue again in ascending ethanol immersions, followed by two 15-minute immersions in xylene at room temperature. After the last 100%ethanol immersion, mount the sections with 15 microliters of a 5%xylene in neutral gum solution and cover each tissue sample with a cover glass. For automatic whole-slide scanning of the sections, first allow the slides to dry at room temperature for 12 hours to desiccate the tissue samples.Next, in the automatic whole-slide scanner, select Brightfield manually and enter the manual scanning interface. Revise the name of the slides and select a storage path for the images. Set the scanning area and select the scan samples using User-Set Thresholds option.The threshold is about 50 with a scanning-area expansion value of 200 micrometers. Set the scanning focus value and select Automatic Focus and Single Layer mode, then load the slides onto the work station and start the automatic scanning. When all of the slides have been scanned, save the images for later analysis.To analyze the images of the lung tissue sections, open the histopathological image-analysis software and select Local Computer to open the file with the scanning data. Select the appropriate zoom level and use Toggle Color Adjust to set the color to the optimal contrast, then circle and name the regions of interest on each image. Next, under plugins, select QC.The Scenario Builder will pop up.Select Density Quant and adjust the detection bar. Adjust the score levels until the staining intensity of the circled areas is similar to that of the original images, and store and name the model as a new file, then apply the model to other slides and select Selected Annotation to let the software automatically calculate the APE score for the regions of interest in each image. Whole-slide digital scanning of lung tissue sections of tumor and adjacent non-tumor tissues from non-small cell lung cancer specimens produces digital images of a high quality.APE scores calculated from these types of lung cancer samples are typically significantly higher than those determined for the adjacent non-cancer tissues. In addition, ZWINT expression levels in squamous-cell carcinoma tissue sections are frequently determined to be significantly higher than those measured in abnocarcinoma tissue samples. While attempting this procedure, it's important to remember to prepare with high quality of the tumor or tumor areas during making pathological scanning.Following this procedure, other methods of the microscopy can be performed in order to answer additional questions. Neither the or different method for pathological scanning. After this technique paved the way for researchers in the field of pathological scanning to explore digital diagnostics in the research purpose.Don't forget that working with paraformaldehyde can be extremely hazardous and precautions such as masks should always be taken while performing this procedure.

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Research

JoVE Journal

Cancer Research

5.3K Views.  Zhongnan Hospital of Wuhan University. This method can help answer key questions in the pathological and the immunohistochemistry. The main advantage of this technique is that faster enough and that produces digital media with high quality. Begin by fixing a three-by-three centimeter kung tissue sample in 4%paraformaldehyde for 24 hours at room temperature.The next day, dehydrate the tissue in ascending ethanol immersions at room temperature, followed by three immersions in 100%xylene for 20 minutes per treatment. After the...