Name: quantitation of protein expression and co-localization using multiplexed immuno-histochemical staining and multispectral imaging
Uploaded: 2016-04-08T15:00:00
Description: Watch this Scientific Journal Video about Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging at JoVE.com

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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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>
</table>

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...

Watch this Scientific Journal Video about Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging at JoVE.com

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 ...

Research

JoVE Journal

Biology

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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

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