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>
	<li>Valdman, A., 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+redox+pathway+enzymes+in+human+prostatic+tissue.">Expression of redox pathway enzymes in human prostatic tissue.</a> Anal Quant Cytol Histol. 31 (6), 367-374 (2009).</li><li>Rimm, D. L., Camp, R. L., Charette, L. A., Olsen, D. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+analyses.+Correlation+of+biochemical+and+immunohistochemical+methods+using+monoclonal+antireceptor+antibodies.">Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies.</a> Arch Pathol Lab Med. 109 (8), 716-721 (1985).</li><li>Volante, 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=Somatostatin+receptor+type+2A+immunohistochemistry+in+neuroendocrine+tumors:+a+proposal+of+scoring+system+correlated+with+somatostatin+receptor+scintigraphy.">Somatostatin receptor type 2A immunohistochemistry in neuroendocrine tumors: a proposal of scoring system correlated with somatostatin receptor scintigraphy.</a> Mod Pathol. 20 (11), 1172-1182 (2007).</li><li>Muris, J. 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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-labeled+antibodies:+preparation+and+application+for+the+localization+of+antigens.">Enzyme-labeled antibodies: preparation and application for the localization of antigens.</a> J Histochem Cytochem. 14 (12), 929-931 (1966).</li><li>Peters, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+tissue+organelles+by+a+combination+of+analytical+subcellular+fractionation+and+enzymic+microanalysis:+a+new+approach+to+pathology.">Investigation of tissue organelles by a combination of analytical subcellular fractionation and enzymic microanalysis: a new approach to pathology.</a> J Clin Pathol. 34 (1), 1-12 (1981).</li><li>Emmert-Buck, M. R., 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=Laser+Capture+Microdissection.">Laser Capture Microdissection.</a> Science. 274 (5289), 998-1001 (1996).</li><li>Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+and+precision+in+quantitative+fluorescence+microscopy.">Accuracy and precision in quantitative fluorescence microscopy.</a> J Cell Biol. 185 (7), 1135-1148 (2009).</li><li>Huang, W., Hennrick, K., Drew, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorful+future+of+quantitative+pathology:+validation+of+Vectra+technology+using+chromogenic+multiplexed+immunohistochemistry+and+prostate+tissue+microarrays.">A colorful future of quantitative pathology: validation of Vectra technology using chromogenic multiplexed immunohistochemistry and prostate tissue microarrays.</a> Hum Pathol. 44 (1), 29-38 (2013).</li><li>Rimm, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-Path:+A+Watson-Like+Visit+to+the+Pathology+Lab.">C-Path: A Watson-Like Visit to the Pathology Lab.</a> Science Translational Medicine. 3 (108), (2011).</li><li>Fiore, C., 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=Utility+of+multispectral+imaging+in+automated+quantitative+scoring+of+immunohistochemistry.">Utility of multispectral imaging in automated quantitative scoring of immunohistochemistry.</a> J Clin Pathol. 65 (6), 496-502 (2012).</li><li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunohistochemistry,+imaging,+and+quantitation:+A+review,+with+an+assessment+of+Tyramide+signal+amplification,+multispectral+imaging+and+multiplex+analysis.">Multiplexed immunohistochemistry, imaging, and quantitation: A review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70 (1), 46-58 (2014).</li><li>Rizzardi, A. E., 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=Quantitative+comparison+of+immunohistochemical+staining+measured+by+digital+image+analysis+versus+pathologist+visual+scoring.">Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring.</a> Diagn Pathol. 7, 42 (2012).</li><li>Bauman, T. 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=Characterization+of+fibrillar+collagens+and+extracellular+matrix+of+glandular+benign+prostatic+hyperplasia+nodules.">Characterization of fibrillar collagens and extracellular matrix of glandular benign prostatic hyperplasia nodules.</a> PLoS One. 9 (10), e109102 (2014).</li><li>Bauman, T. 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=Beta-catenin+is+elevated+in+human+benign+prostatic+hyperplasia+specimens+compared+to+histologically+normal+prostate+tissue.">Beta-catenin is elevated in human benign prostatic hyperplasia specimens compared to histologically normal prostate tissue.</a> Am J Clin Exp Urol. 2 (4), 313-322 (2014).</li><li>Bauman, T. M., Ewald, J. A., Huang, W., Ricke, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD147+expression+predicts+biochemical+recurrence+after+prostatectomy+independent+of+histologic+and+pathologic+features.">CD147 expression predicts biochemical recurrence after prostatectomy independent of histologic and pathologic features.</a> BMC Cancer. 15 (1), 549 (2015).</li><li>Bauman, T. 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=Finasteride+treatment+alters+tissue+specific+androgen+receptor+expression+in+prostate+tissues.">Finasteride treatment alters tissue specific androgen receptor expression in prostate tissues.</a> Prostate. 74 (9), 923-932 (2014).</li><li>Nicholson, T. M., Sehgal, P. D., Drew, S. A., Huang, W., Ricke, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+steroid+receptor+expression+and+localization+in+benign+prostatic+hyperplasia+varies+with+tissue+compartment.">Sex steroid receptor expression and localization in benign prostatic hyperplasia varies with tissue compartment.</a> Differentiation. 85 (4-5), 140-149 (2013).</li><li>Taylor, C. R., Levenson, R. M. <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+immunohistochemistry--issues+concerning+methods,+utility+and+semiquantitative+assessment+II.">Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II.</a> Histopathology. 49 (4), 411-424 (2006).</li><li>Matos, L. L., Trufelli, D. C., de Matos, M. G., da Silva Pinhal, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+as+an+important+tool+in+biomarkers+detection+and+clinical+practice.">Immunohistochemistry as an important tool in biomarkers detection and clinical practice.</a> Biomark Insights. 5, 9-20 (2010).</li><li>Kononen, 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=Tissue+microarrays+for+high-throughput+molecular+profiling+of+tumor+specimens.">Tissue microarrays for high-throughput molecular profiling of tumor specimens.</a> Nat Med. 4 (7), 844-847 (1998).</li><li>Ong, C. 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=Computer-assisted+pathological+immunohistochemistry+scoring+is+more+time-effective+than+conventional+scoring,+but+provides+no+analytical+advantage.">Computer-assisted pathological immunohistochemistry scoring is more time-effective than conventional scoring, but provides no analytical advantage.</a> Histopathology. 56 (4), 523-529 (2010).</li></ol>

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 ...

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.

quantitation-protein-expression-co-localization-using-multiplexed

Research

JoVE Journal

Biology

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

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

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...