Multiplex spatial protein detection by combining immunofluorescence with immunohistochemistry Journal Article
| Authors: | Kang, W.; Santella, A.; Rosiek, E.; Pulina, M.; Chan, E.; Fan, N.; Tipping, M. J.; Barlas, A.; Romin, Y.; Manova-Todorova, K. |
| Article Title: | Multiplex spatial protein detection by combining immunofluorescence with immunohistochemistry |
| Abstract: | Technologies for staining and imaging multiple antigens in single tissue sections are developing rapidly due to their potential to uncover spatial relationships between proteins with cellular resolution. Detections are performed simultaneously or sequentially depending on the approach. However, several technologies can detect limited numbers of antigens or require expensive equipment and reagents. Another serious concern is the lack of flexibility. Most commercialized reagents are validated for defined antibody panels, and introducing any changes is laborious and costly. In this chapter, we describe a method where we combine, for the first time, multiplexed IF followed by sequential immunohistochemistry (IHC) with AEC chromogen on Leica Bond staining processors with paraffin tissue sections. We present data for successful detection of 10 antigens in a single tissue section with preserved tissue integrity. Our method is designed for use with any combination of antibodies of interest, with images collected using whole slide scanners. We include an image viewing and image analysis workflow using nonlinear warping to combine all staining passes in a single full-resolution image of the entire tissue section, aligned at the single cell level. © 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature. |
| Keywords: | immunohistochemistry; unclassified drug; nonhuman; proteins; protein analysis; mouse; metabolism; animal tissue; image analysis; glial fibrillary acidic protein; protein; immunofluorescence; molecular imaging; uvomorulin; tumor marker; tissue section; fluorescent antibody technique; antigen; antigens; staining; transcription factor erg; cytokeratin 5; visualization; cell level; antibody; staining and labeling; collagen type 4; cytokeratin 18; cytokeratin 8; paraffin embedding; single cell analysis; transcription factor sox9; peptides and proteins; multiplex; alignment; workflow; immunohistochemistry (ihc); biomarkers, tumor; immunofluorescence (if); leica bond rx; platelet endothelial cell adhesion molecule 1; desmin (protein); aec chromogen; b220 protein; iba1 protein |
| Journal Title: | Methods in Molecular Biology |
| Volume: | 2593 |
| ISSN: | 1064-3745 |
| Publisher: | Humana Press Inc |
| Date Published: | 2023-01-01 |
| Start Page: | 233 |
| End Page: | 244 |
| Language: | English |
| DOI: | 10.1007/978-1-0716-2811-9_15 |
| PUBMED: | 36513935 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Book Chapter: 15 in "Signal Transduction Immunohistochemistry: Methods and Protocols, 3rd edition" (ISBN: 978-1-0716-2810-2) -- The MSK Cancer Center Support Grant (P30 CA008748) is acknowledged in the PDF -- Source: Scopus |
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