The authors thank the University of Wisconsin Translational Research Initiatives in Pathology laboratory, in part supported by the UW Department of Pathology and Laboratory Medicine and UWCCC grant P30 CA014520, for use of its facilities and services.

&#160; &#160; &#160;NOTE: The protocol herein describes staining and analysis of a tissue microarray (TMA), described previously 12,17,18. The 4 &#181;m thick TMA section was obtained from a paraffin block using a standard microtome.&#160;
&#160; &#160; &#160;NOTE: A spectral library for the 4 chromogens and counterstain should be created for image quantitation. In order to do this, the optimized protocol for each individual antibody should be run with one antibody per slide, minus the final counterstain. A fifth slide shou...

<ol>
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A., Provost, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+tissue+by+construction+of+tissue+microarrays.">Amplification of tissue by construction of tissue microarrays.</a> Exp Mol Pathol. 70 (3), 255-264 (2001).</li><li>Jonmarker, S., 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=Expression+of+PDX-1+in+prostate+cancer,+prostatic+intraepithelial+neoplasia+and+benign+prostatic+tissue.">Expression of PDX-1 in prostate cancer, prostatic intraepithelial neoplasia and benign prostatic tissue.</a> APMIS. 116 (6), 491-498 (2008).</li><li>McCarty, K. S., Miller, L. S., Cox, E. B., Konrath, J., McCarty, K. 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J., 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=Immunohistochemical+profiling+of+caspase+signaling+pathways+predicts+clinical+response+to+chemotherapy+in+primary+nodal+diffuse+large+B-cell+lymphomas.">Immunohistochemical profiling of caspase signaling pathways predicts clinical response to chemotherapy in primary nodal diffuse large B-cell lymphomas.</a> Blood. 105 (7), 2916-2923 (2005).</li><li>Jaraj, S. J., 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=Intra-+and+interobserver+reproducibility+of+interpretation+of+immunohistochemical+stains+of+prostate+cancer.">Intra- and interobserver reproducibility of interpretation of immunohistochemical stains of prostate cancer.</a> Virchows Arch. 455 (4), 375-381 (2009).</li><li>Nakane, P. K., Pierce, G. 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The use of traditional immunohistochemistry for evaluating protein expression is limited by subjective, semi-quantitative methods of analysis 22,23. Advance platforms have been created for high-throughput analysis of biomarker expression and localization. Detailed segmentation of both tissue and subcellular compartments allows users to study biomarker expression, localization, and co-localization with other markers of interest. In previous studies, we have demonstrated the utility of IHC and multispectral imag...

Immunohistochemistry (IHC) is a standard lab technique for detection of protein within tissue, and IHC is still widely used in both research and diagnostic pathology. The evaluation of IHC staining is often semi-quantitative, introducing potential bias into interpretation of results. Many semi-quantitative approaches have been developed which incorporate both staining intensity and staining extent into final diagnosis 1-4. Other systems include scoring intensity and subcellular location in order to better localize expression 5. Incorporation of average scores from multiple viewers is often utilized in order to minimize the effects of single viewer bias 6. Despite these efforts, subjectivity in analysis still remains, particularly when evaluating the extent of staining 7. Protocol standardization and minimizing subjectivity from human input is paramount to creating accurate, reproducible IHC results.
There are other options besides IHC for determining protein expression within tissues. Within the research setting, immunohistochemistry has traditionally been viewed as a means to examine protein localization 8, while other techniques such as immunoblotting are viewed as gold standard for investigating protein expression. Determining tissue or cell compartment-specific expression is difficult without incorporating advanced techniques such as cell fractionation or laser capture microdissection 9,10. The use of fluorescent antibodies on tissue slides offers a reasonable compromise, but background autofluorescence due to NADPH, lipofuscins, reticular fibers, collagen, and elastin can make accurate quantitation difficult 11.
Automated computational pathology platforms are a promising direction for more objective quantitation of pathology staining 12-15. Combining multispectral imaging with tissue microarrays facilitates high-throughput analysis of protein expression in large sample sizes. With these techniques, analysis of protein co-localization, staining heterogeneity, and tissue and subcellular localization is possible while substantially reducing both inherent biases and time necessary for analysis, while returning data in a continuous rather than categorical format 16. Therefore, the purpose of this study was to demonstrate the utility of and methodology for performing multiplexed immunohistochemistry with analysis, using multispectral imaging software.
This protocol is written for manual, multiplex immunohistochemical staining of a single tissue section slide with four optimized monoclonal antibodies. As a representative experiment, nuclear anti-rabbit estrogen receptor alpha (ER&#945;) and androgen receptor (AR) are multiplexed with membrane-bound anti-mouse CD147 and membrane-bound anti-mouse E-cadherin. Any antibody of choice may be substituted for the antibodies listed herein, but each combination of antibodies requires separate optimization. Pre-treatment for all the antibodies must be identical. The AR and CD147 antibodies should be optimized individually and then as a cocktail. Each antibody is detected using a biotin-free polymer system and one of 4 unique chromogens.

<table border="0" cellpadding="0" cellspacing="0" width="945">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylene</td>
			<td style="width:127px;">Fisher Chemical</td>
			<td style="width:119px;">X3F1GAL</td>
			<td style="width:483px;">NFPA rating:Health &#8211; 2, Fire &#8211; 3 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethyl Alcohol-200 proof</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">4355223</td>
			<td>NFPA rating:Health &#8211; 0, Fire &#8211; 3 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris Base</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-500</td>
			<td>NFPA rating:Health &#8211; 2, Fire &#8211; 0 , Reactivity-0</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris Hydroxymethyl aminomethane HCl</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">BP153-1</td>
			<td>NFPA rating:Health &#8211; 2, Fire &#8211; 0 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20</td>
			<td style="width:127px;">Chem-Impex</td>
			<td style="width:119px;">1512</td>
			<td>NFPA rating:Health &#8211; 0, Fire &#8211;1 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate-buffered saline</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">BP2944-100</td>
			<td>NFPA rating:Health &#8211; 1, Fire &#8211;0 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peroxidazed</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">PX968</td>
			<td>Avoid contact with skin and eyes. May cause skin irritation and eye damage.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Diva Decloaker&#160;</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">DV2004</td>
			<td style="width:483px;">This product has been classified as non&#8208;hazardous based on the physical&#160; and/or&#160; chemical nature and/or concentration of ingredients.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Estrogen Receptor alpha</td>
			<td style="width:127px;">Thermo Fisher Scientific-Labvision</td>
			<td style="width:119px;">RM9101</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Androgen Receptor</td>
			<td style="width:127px;">SCBT</td>
			<td style="width:119px;">sc-816</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD147</td>
			<td style="width:127px;">Biodesign</td>
			<td style="width:119px;">P87535M</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">E-cadherin</td>
			<td style="width:127px;">Dako</td>
			<td style="width:119px;">M3612</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Renoir Red Andibody Diluent</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">PD904</td>
			<td>It is specially designed to work with Tris-based antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DeCloaking Chamber&#160;</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">Model DC2002</td>
			<td>Take normal precautions for using a pressure cooker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Barrier pen-Immuno Edge&#160;</td>
			<td style="width:127px;">Vector Labs</td>
			<td style="width:119px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Denaturing Kit-Elution step</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">DNS001H</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mach 2 Goat anti-Rabbit HRP Polymer</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">RHRP520</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mach 2 Goat anti-Rabbit AP Polymer</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">RALP525</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mach 2 Goat anti-Mouse HRP Polymer</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">M3M530</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Betazoid DAB Chromogen Kit</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">BDB2004</td>
			<td style="width:483px;">1. DAB is known to be a suspected carcinogen. 
			2. Do not expose DAB components to strong light or direct sunlight. 
			3. Wear appropriate personal protective equipment and clothing. 
			4. DAB may cause sensitization of skin. Avoid contact with skin and eyes. 
			5. Observe all federal, state and local environmental regarding disposal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Warp Red Chromogen Kit</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">WR806</td>
			<td style="width:483px;">Corrosive. Acid that may cause skin irritation or eye damage.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vina Green Chromogen Kit</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">BRR807</td>
			<td>Harmful if swallowed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bajoran Purple Chromogen Kit</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">BJP807</td>
			<td>Flammable liquid. Keep away from heat, flames and sparks. Harmful by ingestion or absorption. Avoid contact with skin or eyes, and avoid inhalation.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Cat Hematoxylin</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">CATHE</td>
			<td style="width:483px;">Purple &#160;solution &#160;with &#160;a &#160;mild &#160;acetic &#160;acid &#160;(vinegar) &#160;scent. &#160;May &#160;be 
			&#160;irritating &#160;to &#160;skin &#160;or &#160;eyes. &#160;Avoid &#160;contact &#160;with &#160;skin &#160;and &#160;eyes. &#160;Avoid &#160;ingestion.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">XYL Mounting Media</td>
			<td style="width:127px;">Richard Allen</td>
			<td style="width:119px;">8312-4</td>
			<td>NFPA rating:Health &#8211; 2, Fire &#8211; 3 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 Coverslips</td>
			<td style="width:127px;">Fisher Brand</td>
			<td align="right" style="width:119px;">22266858</td>
			<td>Sharp edges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubation (Humidity)Chamber</td>
			<td style="width:127px;">obsolete</td>
			<td style="width:119px;">obsolete</td>
			<td>Multiple vendors available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Convection Oven</td>
			<td style="width:127px;">Stabil- Therm</td>
			<td style="width:119px;">C-4008-Q</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Background Punisher Blocking Reagent</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">BP974</td>
			<td>This product is not classified as hazardous.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">inForm software</td>
			<td style="width:127px;">PerkinElmer</td>
			<td style="width:119px;">CLS135781</td>
			<td style="width:483px;">Primary multispectral imaging software used in manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuance software</td>
			<td style="width:127px;">PerkinElmer</td>
			<td style="width:119px;">NUANCEEX</td>
			<td>Software used for making spectral libraries within manuscript</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectra microscope and slide scanner</td>
			<td style="width:127px;">PerkinElmer</td>
			<td style="width:119px;">VECTRA</td>
			<td>Automated slide scanner and microscope for obtaining IM3 image cubes</td>
		</tr>
	</tbody>
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quantitation of protein expression and co-localization using multiplexed immuno-histochemical staining and multispectral imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

In Figure 1, training is performed on prostate tissues to segment images into epithelial and stromal portions, along with the non-tissue compartment. By using the epithelial membrane marker E-cadherin, cell segmentation was performed to separate the nucleus, cytoplasm, and membrane portions, shown in Figure 2.
In one experiment, we used multiplexed IHC to investigate the expression and localizat...

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Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

The overall goal of this method is to objectively quantitate protein expression and co-localization using multispectral imaging. This method can help answer key questions in the basic research and diagnostic pathology field, such as how proteins change expression and localization after treatment, or in disease progression. The main advantage of this technique is that it removes the operator subjectivity inherent to traditional quantification methods.To begin the quantification process, open the multispectral imaging software to build a spectral library from previously prepared control slides stained with individual chromogens and the hematoxylin stained slide. Next, open an image cube acquired from a control slide, and select four to five positively stained areas to optically define the chromogen. Repeat the steps with image cubes from other control slides until a complete spectral library representing all chromogens is created, and then save the spectral library.Begin a new project within the multispectral imaging software by selecting Multispectral or im3 for the image format option, and Brightfield for the sample format. Configure the project by choosing segment tissue, find features, phenotyping, score, and export. Change the image resolution to expedite the analysis time, if desired.Import the previously created spectral library and select all chromogens to be included in the analysis. Open the image cubes to be included in the training data set by selecting the Open Image Cube option. Select at least 18%of the total number of images to be analyzed to ensure training accuracy.Next, choose the training set of images. Images that represent all disease states, to increase segmentation accuracy. Include abundant negative staining images in the training set to avoid bias during this step.White balance the images in the training set by selecting the eye dropper tool, and choosing an area in one image that is white. Select the advance button to move tissue segmentation. Then use the tissue categories panel to choose the tissue types to be analyzed for more accurate protein tissue localization select tissue categories can be used.Begin creating the algorithm and defining tissue categories by using the pen tool and drawing around groups of cells within training images. When finished with one tissue category repeat the step for other tissue categories. Be sure to choose groups of cells within images that are characteristic of the tissue category type.Select components to be included in training for the tissue segmenter. Choose an appropriate pattern scale to train the tissue segmenter. Then select the trained tissue segmenter button.Observe a pop up box displaying accuracy of the proportion of pixels within properly classified training regions. Segment the entire training set of images by clicking segment images. When finished review the training set to find any misclassified tissue with a current training algorithm.When confident with tissue segmentation algorithm results select the advance button. Ensure that nuclei is already selected to choose cytoplasm and/or membrane. Select the nuclei tab and choose the appropriate settings for nuclear segmentation.Then choose whether individual or all tissue categories will be included in segmentation. Select the counterstain object based threshold approach for a simplified method to obtain good results. Next, select the object based threshold approach on a reliable nuclear counterstain.Select cytoplasm shaped parameters by choosing the cytoplasm tab. Next, select outer distance to nucleus. Then select minimum size.Select the next option component with primary, secondary and select tertiary as secondary options. Move to the membrane tab. Choose first for the membrane specific marker used.Adjust the full scale optical density or OD to find a minimum threshold or a positive for each marker on cell membranes. Continuing under the membrane tab select the maximum cell size. After selecting all of the options select segment all.Apply the settings to the images and observe them. Choose advance to move to the score IHC step and choose a tissue category to score. Choose a desired scoring type and choose the cell compartment to be used in the scoring analysis.Next, select view component data and move the cursor over the training images to find appropriate optical density minimal threshold of staining for positive cells for the components of interest. Export the data for the training set to test the algorithm created through tissue segmentation cell segmentation and scoring values follow the prompts to create a new folder for the export directory. Select images and tables to be created and included in analysis.Next, perform the analysis by selecting export for all. When the analysis is complete, view the cell segmentation and scoring data for images with both high and low staining to evaluate the accuracy of the settings. Once satisfied with the settings, click on the batch analysis tab to copy the algorithm to the active project.Then choose a new export directory and select the images and tables to include in the analysis. Under the input files option choose all images to be included in the batch analysis. Select the run option to perform the analysis.When completed advance to the review merge tab. Select include all and select merge to create data sheets with summary data for analysis. Training was performed on prostate tissues to segment images into epithelial and stromal portions.Along with a non-tissue compartment. A set of training images were imported into multispectral imaging software representing tissue types and disease states of the entire set of images. Tissue categories were created including stroma, epithelia and non-tissue and categories were defined by manually drawing on top of training images.An algorithm for tissue segmentation was created and applied to the training set of images. Accurately segmenting the tissues. Using the multiplex immunohistochemistry technique cells positive for nuclear expression of ER-alpha seen as red and AR seen as brown were identified despite overlapping color and metric signals The cell membrane specific expression of CD-147 was quantified by using E-catherine seen as black as a marker protein.After watching this video you should have a good understanding of how to quantitate protein expression and co-localization inform and fix paraffin embedded tissues.

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12.6K vistas.  Washington University in St. Louis School of Medicine. The overall goal of this method is to objectively quantitate protein expression and co-localization using multispectral imaging. This method can help answer key questions in the basic research and diagnostic pathology field, such as how proteins change expression and localization after treatment, or in disease progression. The main advantage of this technique is that it removes the operator subjectivity inherent to traditional quantification methods.To begin the quantification process,...