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To tackle these issues, here we report the development of a highly sensitive and multiplexed in situ protein profiling method using off-the-shelf antibodies. In this approach, the protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and cleavable fluorophores via click chemistry. Through reiterative cycles of target staining, fluorescence imaging, and fluoropohore cleavage, many proteins can be profiled in single cells in situ. Applying this approach, we successfully quantified 28 different proteins in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest multiplexing capacity among the tyramide signal amplification (TSA) methods. Based on their unique protein expression patterns and their microenvironment, \u223c820,000 cells in the tissue are classified into distinct cell clusters. We also explored the cell-cell interactions between these varied cell clusters and observed different subregions of the tissue are composed of cells from specific clusters.<\/jats:p>","DOI":"10.1101\/2023.10.20.563323","type":"posted-content","created":{"date-parts":[[2023,10,23]],"date-time":"2023-10-23T23:30:28Z","timestamp":1698103828000},"source":"Crossref","is-referenced-by-count":0,"title":["Ultrasensitive and multiplexed protein imaging with clickable and cleavable fluorophores"],"prefix":"10.64898","author":[{"given":"Thai","family":"Pham","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joshua","family":"Labaer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jia","family":"Guo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023102708151296000_2023.10.20.563323v1.1","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.cels.2016.03.008","article-title":"Multiplexed Epitope-Based Tissue Imaging for Discovery and Healthcare Applications","volume":"2","year":"2016","journal-title":"Cell Systems"},{"key":"2023102708151296000_2023.10.20.563323v1.2","doi-asserted-by":"crossref","first-page":"7083","DOI":"10.1002\/chem.201705014","article-title":"Highly Multiplexed Single-Cell Protein Analysis","volume":"24","year":"2018","journal-title":"Chemistry - A European Journal"},{"key":"2023102708151296000_2023.10.20.563323v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/nrg3356"},{"key":"2023102708151296000_2023.10.20.563323v1.4","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.200300592"},{"key":"2023102708151296000_2023.10.20.563323v1.5","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1017349108"},{"key":"2023102708151296000_2023.10.20.563323v1.6","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1021\/acsmeasuresciau.2c00011","article-title":"Multiplexed Single-Cell in Situ Protein Profiling","volume":"2","year":"2022","journal-title":"ACS Meas Sci Au"},{"key":"2023102708151296000_2023.10.20.563323v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1250"},{"key":"2023102708151296000_2023.10.20.563323v1.8","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkr1209"},{"key":"2023102708151296000_2023.10.20.563323v1.9","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201204304"},{"key":"2023102708151296000_2023.10.20.563323v1.10","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1300136110"},{"key":"2023102708151296000_2023.10.20.563323v1.11","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms9390"},{"key":"2023102708151296000_2023.10.20.563323v1.12","doi-asserted-by":"crossref","first-page":"2636","DOI":"10.1002\/anie.201611641","article-title":"Highly Multiplexed Single-Cell In Situ Protein Analysis with Cleavable Fluorescent Antibodies","volume":"56","year":"2017","journal-title":"Angewandte Chemie International Edition"},{"key":"2023102708151296000_2023.10.20.563323v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2018.07.010"},{"key":"2023102708151296000_2023.10.20.563323v1.14","doi-asserted-by":"publisher","DOI":"10.1126\/science.aar7042"},{"key":"2023102708151296000_2023.10.20.563323v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/nm.3488"},{"key":"2023102708151296000_2023.10.20.563323v1.16","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1038\/nmeth.2869","article-title":"Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry","volume":"11","year":"2014","journal-title":"Nature methods"},{"key":"2023102708151296000_2023.10.20.563323v1.17","doi-asserted-by":"crossref","unstructured":"Liao, R. , Mondal, M. , Nazaroff, C. 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