Start • Life Sciences And Research Solutions • Modern Multiplex Solutions for the Research Lab Modern Multiplex Solutions for the Research Lab Image Multiplexing addresses the need for researchers to assess multiple biomarkers (protein and/or nucleic acid markers) at specific locations within a tissue sample. The information revealed through simultaneous detection of multiple markers, the spatial relationships among cells and tissue in disease, and the heterogeneity are now understood to be critical to developing effective therapeutic strategies. The latest technology encompasses multiplex IHC as well as multiplex ISH and FISH. View whole slide images Key considerations for choosing to Multiplex The need to extract the maximum amount of data from a limited sample, multiplex technology enables the user to detect many biomarkers in a single tissue section. Multiplexing can help determine which targets are important, by starting with a large range of potential markers and using the resulting spatial data to refine to the critical few. Multiplex staining on tissue provides cell-specific context that molecular techniques and PCR can’t provide. Tissue multiplexing visualizes both protein and nucleic acid targets in the same tissue section. Image Choosing the right tool to help answer your research question The Fundamentals The Difference Between Number of Colors, Plex, and Multiplexing Number of colors: Every single stain on the slide including counterstains DAPI (counterstain) CY7 Spectrum Green Spectrum Orange Plex: The number of targets to be analyzed excluding counterstain Dual or 2-plex red and brown IHC with crystal light green counterstain Dual or 2-plex red and green RNA ISH with hematoxylin counterstain Multiplexing: the ability to simultaneously detect 2 or more markers on a single slide (eg CD3, CD4, CD8 & counterstain). The nuclear counterstains most frequently used are hematoxylin for brightfield and DAPI for fluorescent microscopy Ultivue PD-L1 kit staining human lung, 4-plex plus DAPI (The protocol was carried out on the BOND RX fully automated stainer from Leica Biosystems and the stained tissue imaged on the Aperio VERSA whole slide scanning system) Fluorescence Multiplexing Rat Neuron, 6-plex Spectrum Green DAPI Spectrum Orange Texas Red CY7 CY5 Aqua Ultivue PD-L1 kit staining human lung, 4-plex plus DAPI (The protocol was carried out on the BOND RX fully automated research stainer and the stained tissue imaged on the Aperio VERSA whole slide scanning system) Chromogenic Multiplexing Chromogenic Multiplexing (multiplex IHC and ISH) Chromogenic multiplexing provides the ability to look at 3 or more markers on the same slide using brightfield microscopy. Chromogens provide a more stable and permanent result compared to their fluorescent counterparts. Image Normal human tonsil stained with: Marker 1 - CyclinD1 - Red Marker 2 - CD20 - Brown Counterstain = Hematoxylin Image Normal human tonsil stained with: Cyclin D1 red nuclear Image Normal human tonsil stained with: CD20 brown membranous stain Image Normal human tonsil stained with: Hematoxylin counterstain Image Normal tonsil tissue stained with: Marker 1 - PD-L1 - Red Marker 2 - CD68 - DAB Marker 3 - CD8 - Blue Marker 4 - Pan-CK - Green Counterstain = Hematoxylin Image Melanoma in skin stained with: Marker 1 - PD-L1 - Red Marker 2 - CD68 - DAB Marker 3 - CD8 - Blue Marker 4 - Pan-CK - Green Image Melanoma in skin stained with: Marker 1 - PD-L1 - Red Marker 2 - CD68 - DAB Marker 3 - CD8 - Blue Marker 4 - Pan-CK - Green Image Human bladder stained with: Marker 1 - CK20 - Red Marker 2 - p53 - Brown Marker 3 - CD44 - Green Counterstain = Hematoxylin Image Adenocarcinoma in human colon stained with: Marker 1 - CDX2 - Red Marker 2 - CD3 - Green Counterstain = Hematoxylin Explore Our Chromogenic Multiplexing Detection Portfolio Multiple Use Cases Image Immuno-Oncology Get the full picture for your tumor microenvironment research Immuno-oncology has been one of the primary drivers of the current multiplex technology development. Multiplex immunohistochemical (IHC) analysis of formalin-fixed, paraffin-embedded (FFPE) tissue samples enables researchers to study the spatial relationships between different cell phenotypes in situ. Tonsil is often the first step to check that the antibodies are identifying the appropriate immune cells. After that, the actual tumor microenvironment can be visualized with hematologic samples, staining often determines cell lineage/origin as well. This phased process of optimization is long but ensures fidelity of results. Fluorescence In Situ Hybridization (FISH) Assay Nucleic Acids in Cancer research FISH is a sensitive, accurate, and reliable technique widely applied in cancer research. The genetic defects uncovered by FISH represent early genetic triggers or events responsible for cancer at stem-cell level. FISH provides cell-based context for specific genomic aberrations and plays an important role in detecting specific biomarkers in solid and hematologic neoplasms Image Quantitative Multicolor QM- FISH Pairs of probes have been conventionally used to detect a single genetic event like deletion or amplification of a locus or chromosomal translocation. However, with the discovery of multigenic diseases including cancer, simultaneous detection of such genes by using multiple probes on a single slide aids understanding of disease progression (quantitative multicolor FISH). Tissue Heterogeneity It is easy to think of heterogeneity between tumor and non-tumor tissue, but it is also well known that there is heterogeneity within the tumor itself. Not every cell will stain the same and not every marker will be present in the same cells. Multiplex IHC and ISH uncover this multi-faceted approach to tissue heterogeneity and provide context. Example of breast cancer image using Aperio Image Analysis software. Image Educational Resources Image Tips & Tricks to Multiplexing: How to Choose Chromogen Colors for Multiplex and Detection Systems for Multiplex Assays View Article Image Tips & Tricks to Multiplexing: Top 5 Reasons to Multiplex and Chromogenic versus Immunofluorescent Detection View Article Image Selecting the right detection for your IHC/ISH project (Fluorescence vs Chromogenic Staining) View Article Featured Products Image BOND RX Find Breakthroughs Faster with the Freedom to Discover LEARN MORE Image BOND RXm Helping academic researchers achieve more: BOND RXm – Fully Automated Advanced Staining LEARN MORE Image Aperio GT 450 Fast-Forward to Next Generation Imaging LEARN MORE Image Aperio VERSA Delivering Excellence in IHC, ISH and Fluorescent Tissue-Based Research including Multiplex Whole Slide Scanning LEARN MORE For Research Use Only. Not for use in diagnostic procedures. 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