title: Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging.
abstract: 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.
authors.affiliation: Division of Urologic Surgery, Washington University in St. Louis School of Medicine; Department of Urology, University of Wisconsin School of Medicine and Public Health.
authors.forename: Tyler M
authors.initials: TM
authors.lastname: Bauman
authors.affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Ricke
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health.
authors.forename: Sally A
authors.initials: SA
authors.lastname: Drew
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health; O'Brien Urology Research Center, University of Wisconsin School of Medicine and Public Health.
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health; O'Brien Urology Research Center, University of Wisconsin School of Medicine and Public Health; rickew@urology.wisc.edu.
authors.forename: William A
authors.initials: WA
authors.lastname: Ricke
mesh_terms: D000465:Algorithms; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D015203:Reproducibility of Results; D012984:Software; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D011506:Proteins