- [Resource Hub](/) - [Resource](/?resource-type=poster#library) - Introduction of a robust workflow for the whole-slide acquisition and co-registration of multiplex immunofluorescence tissue images for analysis of a 9-color, 8-marker immunophenotyping assay # Introduction of a robust workflow for the whole-slide acquisition and co-registration of multiplex immunofluorescence tissue images for analysis of a 9-color, 8-marker immunophenotyping assay Profiling multiple cell types in tumor tissue takes more markers than conventional immunohistochemistry can support; this AACR 2020 poster from Indica Labs, RareCyte and Ultivue stains 8 markers in a single run, images them across 2 rounds on a CyteFinder II HT scanner, and co-registers the rounds into one 10-channel whole-slide image for cell-level phenotyping. Presented by Indica Labs / RareCyte / Ultivue. Read or download to see: - Eight markers were stained in one run, then imaged in two rounds of four. The UltiMapper I/O Immuno8 kit stains all 8 antibodies together on a Leica Bond automated stainer and amplifies every barcode in the same run; imaging probes complementary to one set of 4 barcodes are then hybridized and scanned on the RareCyte CyteFinder II HT, dehybridized, and replaced with probes for antibodies 5 to 8 for a second scan, building the 9-color panel of CK, CD3, CD4, FoxP3, CD8a, PD-1, PD-L1 and CD68 alongside DAPI. - The two imaging rounds were co-registered at the cellular level and fused into a single 10-channel image. The ome.tiff files from rounds 1 and 2 were aligned with the Serial Stain Registration tool in HALO 3.1, then fused by the Serial Stain/Section module into one 10-channel slide; overlaying each round’s DAPI channel rendered well-aligned nuclei white and misaligned nuclei blue or yellow, and those misaligned nuclei were seen mainly in areas of tissue disturbed between rounds — necrotic areas where cellular debris rinsed off, and small movements or folds in the tissue. - Nuclear detection was keyed to the second round’s DAPI, and one sample was excluded from the results. The kit stained FFPE colorectal cancer, non-small cell lung cancer and human tonsil control tissue; the HighPlex FL module then segmented individual cells and analyzed nuclear, cytoplasm or membrane biomarkers, with nuclear detection based on the round 2 DAPI signal so that only cells that made it through both rounds of staining were quantified, and Sample Lung 3 was excluded from the results for low antigen retrieval during automated staining. Phenotypes were resolved by marker combination, including cytotoxic T cells (CD3+ and CD8+), regulatory T cells (CD3+, CD4+ and FoxP3+) and macrophages (CD68+). [Download poster](https://cdn.sanity.io/files/s3p6uqps/production/cfaa4c07190add36b82fda74e9eb41511f363aa3.pdf) Your browser doesn’t support embedded PDFs. [Download the poster](https://cdn.sanity.io/files/s3p6uqps/production/cfaa4c07190add36b82fda74e9eb41511f363aa3.pdf) to view it. [Back to all resources](/#library) ## Related resources [Article](https://www.selectscience.net/product-news/measuring-versus-reading-transforming-the-field-of-quantitative-pathology?artid=58733) CyteFinder SelectScience ### Measuring versus reading – transforming the field of quantitative pathology [Read Article](https://www.selectscience.net/product-news/measuring-versus-reading-transforming-the-field-of-quantitative-pathology?artid=58733) [Spec Sheet](https://go.pardot.com/l/455452/2019-11-18/7mzyyz/455452/220141/TS20_103.pdf) CyteFinder ### CyteFinder II HT [View Spec Sheet](https://go.pardot.com/l/455452/2019-11-18/7mzyyz/455452/220141/TS20_103.pdf) [Spec Sheet](https://go.pardot.com/l/455452/2019-11-18/7mzyz2/455452/220143/TS20_104.pdf) CyteFinder ### CyteFinder II [View Spec Sheet](https://go.pardot.com/l/455452/2019-11-18/7mzyz2/455452/220143/TS20_104.pdf) [Publication](https://doi.org/10.1200/JCO.2026.44.16_suppl.5041) CyteFinder Journal of Clinical Oncology 2026 ### Clinical validation of circulating tumor cell synaptophysin and androgen receptor expression in metastatic prostate cancer phenotypes. [View Publication](https://doi.org/10.1200/JCO.2026.44.16_suppl.5041) [Video](https://www.youtube.com/watch?v=_oGC1KRVN2U) CyteFinder 2026 ### Dr. Pashtoon Kasi on the impact of circulating tumor cells on clinical research [Watch Video](https://www.youtube.com/watch?v=_oGC1KRVN2U) [Publication](https://doi.org/10.1038/s41587-026-03099-z) CyteFinder Nature Biotechnology 2026 ### mRNA vaccine immunity is enhanced by hepatocyte detargeting and not dependent on dendritic cell expression [View Publication](https://doi.org/10.1038/s41587-026-03099-z) [Publication](https://pmc.ncbi.nlm.nih.gov/articles/PMC7618831/) CyteFinder Cancer Immunology Research 2026 ### Ex Vivo Immuno-Oncology Platform Reveals Spatial T Cell Infiltration Patterns Linked to ATR Inhibition Responses in High-Grade Serous Ovarian Cancer [View Publication](https://pmc.ncbi.nlm.nih.gov/articles/PMC7618831/) [Publication](https://doi.org/10.1097/pai.0000000000001311) CyteFinder Applied Immunohistochemistry & Molecular Morphology 2026 ### Quantitative Assays for TROP2 Measurement in Breast Cancer and Comparison to H-Score [View Publication](https://doi.org/10.1097/pai.0000000000001311) [Publication](https://pmc.ncbi.nlm.nih.gov/articles/PMC12881594/) CyteFinder Nature Communications 2026 ### Extracellular matrix mediates circulating tumor cell clustering in triple-negative breast cancer metastasis [View Publication](https://pmc.ncbi.nlm.nih.gov/articles/PMC12881594/) [Publication](https://doi.org/10.1016/j.ejso.2025.111357) CyteFinder European Journal of Surgical Oncology 2026 ### Circulating tumor cells predict relapse in head and neck mucosal squamous cell carcinoma [View Publication](https://doi.org/10.1016/j.ejso.2025.111357) [Publication](https://pmc.ncbi.nlm.nih.gov/articles/PMC12805936/) CyteFinder Cancer Research Communications 2026 ### Surface Marker Identification to Capture Live Circulating Tumor Cells in Metastatic Triple-Negative Breast Cancer [View Publication](https://pmc.ncbi.nlm.nih.gov/articles/PMC12805936/) [Publication](https://doi.org/10.1158/1078-0432.CCR-25-0244) CyteFinder Clinical Cancer Research 2025 ### Pre-clinical Modeling of Navtemadlin Pharmacokinetics, Pharmacodynamics, and Efficacy in IDH-wildtype Glioblastoma [View Publication](https://doi.org/10.1158/1078-0432.CCR-25-0244)