PMID- 31502167 OWN - NLM STAT- MEDLINE VI - 2055 TI - Multiplexed Immunohistochemical Consecutive Staining on Single Slide (MICSSS): Multiplexed Chromogenic IHC Assay for High-Dimensional Tissue Analysis. PG - 497-519 LA - eng PT - Journal Article PL - United States TA - Methods Mol Biol JT - Methods in molecular biology (Clifton, N.J.) JID - 9214969 IS - 1940-6029 (Electronic) LID - 10.1007/978-1-4939-9773-2_23 [doi] FAU - Akturk, Guray AU - Akturk G AD - Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. FAU - Sweeney, Robert AU - Sweeney R AD - Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. FAU - Remark, Romain AU - Remark R AD - Innate Pharma, Marseille, France. FAU - Merad, Miriam AU - Merad M AD - Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. FAU - Gnjatic, Sacha AU - Gnjatic S AD - Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. sacha.gnjatic@mssm.edu. IS - 1064-3745 (Linking) RN - 0 (Biomarkers, Tumor) RN - 0 (Chromogenic Compounds) SB - IM MH - Biomarkers, Tumor/*metabolism MH - Chromogenic Compounds/chemistry MH - Humans MH - Immunohistochemistry MH - Machine Learning MH - Neoplasms/*metabolism MH - Paraffin Embedding MH - Tissue Fixation MH - Tumor Microenvironment OTO - NOTNLM OT - *Biomarkers OT - *CD20 OT - *CD3 OT - *CD66b OT - *CD68 OT - *CD8 OT - *Cancer immunotherapy OT - *Cell segmentation OT - *Chromogenic immunohistochemistry OT - *Consecutive staining OT - *FOXP3 OT - *Histology OT - *Image analysis OT - *Immuno-oncology OT - *Immunostaining OT - *In situ markers OT - *Machine learning OT - *Morphology OT - *Multiplexed immunohistochemistry OT - *PD-1 OT - *PD-L1 OT - *Positive cell detection OT - *Random forest OT - *Serial staining OT - *Single slide OT - *Whole slide imaging PMC - PMC7869027 DCOM- 20201109 LR - 20210210 DP - 2020 AB - Disease states and cellular compartments can display a remarkable amount of heterogeneity, and truly appreciating this heterogeneity requires the ability to detect and probe each subpopulation present. A myriad of recent single-cell assays has allowed for in-depth analysis of these diverse cellular populations; however, fully understanding the interplay between each cell type requires knowledge not only of their mere presence but also of their spatial organization and their relation one to the other. Immunohistochemistry allows for the visualization of cells and tissue; however, standard techniques only allow for the use of very few probes on a single specimen, not allowing for in-depth analysis of complex cellular heterogeneity. A number of multiplex imaging techniques, such as immunofluorescence and multiplex immunohistochemistry, have been proposed to allow probing more cellular markers at once; however, many of these techniques still have their limitations. The use of fluorescent markers has an inherent limitation to the number of probes that can be simultaneously used due to spectral overlap. Moreover, other proposed multiplex IHC methods are time-consuming and require expensive reagents. Still, many of the methods rely on frozen tissue, which deviates from standards in human pathological evaluation. Here, we describe a multiplex IHC technique, staining for consecutive markers on a single slide, which utilizes similar steps and similar reagents as standard IHC, thus making it possible for any lab with standard IHC capabilities to perform this useful procedure. This method has been validated and confirmed that consecutive markers can be stained without the risk of cross-reactivity between staining cycles. Furthermore, we have validated that this technique does not lead to decreased antigenicity of subsequent epitopes probed, nor does it lead to steric hindrance.