IHC/ISH Multiplexing

SUBTYPE:Gene ExpressionProtein Expression
APPLICATION & SPECIALITY USES:Biomarker DiscoveryCell Therapy/ Regenerative MedicineImmuno-oncologyPersonalized Medicine
Keywords:Antibody ReplacementAntibody ValidationImmuno-Oncologyin situ HybridizationIO DevelopmentMultiplex in situ HybridizationTarget ValidationVectra Polaris


Our PHASEZERO® Research Services team has established expertise in both ViewRNA® and RNAscope® branched chain in situ hybridization techniques (ISH). Thorough assay development and optimization approaches allow us to effectively use these techniques to deliver high quality, biologically relevant data supporting human tissue based target and biomarker validation projects and diagnostic assay development.

We have developed an approach to allow sequential ISH and IHC in the same human tissue section. The advantage of multiplexing ISH and IHC is to allow use of validated antibodies together with mRNA detection for target localization (e.g. for validation of protein and mRNA expression) and where an appropriate antibody for one of the targets is not available or not trusted. This again is performed in fully-automated environment, providing an increased throughput and reproducibility versus a manual approach.

Using multiplexed approaches such as these has clear application across many areas of disease biology, in particular for novel immuno-oncology approaches where understanding of specific immune cell interactions with solid tumors is paramount.

Features & Benefits

IHC assays are fully automated and run on the following machines:
• Dako Autostainer Plus (IQ/OQ/PQ validated)
• Ventana Discovery XT
• Leica BondRX machines

Facility and Capabilities
• GLP accredited for moderate throughput IHC
• Fully trained, experienced scientists included 3 GLP TCR Study Directors
• Consulatant Histopathologists – Clinical and Veterinary
• SOPs for complete process – Tissue Validation, Preparation, Assay, Analysis and Reporting
• Digital Pathology – Aperio ScanScope AT Turbo Image analysis, archiving and on-line peer review

• Wide experience of multiple detection systems and protocols
• Range of antigen retrieval approaches including high pressure/ high temperature
• Dako Envision, Envision Flex+, and CSAII™;  Vector ABC and ImmPress™; and Menarini polymer HRP detection and fluorescent labeled secondary antibodies
• Alkaline phosphatase, horseradish peroxidase and glucose oxidase chromogenic endpoints
• Ventana Omnimap™ and Ultramap™ detection systems
• Leica bond polymer refine detection
• Multiple antibody species detection available including mouse, rabbit, goat, rat and chicken
• Multiplexing with chromogenic or fluorescent endpoints

• Fluorescence analysis and multiplexing (7 targets simultaneously)
• Confocal and fluorescence microscopy
• Scalable via use of TMAs
• Semi-quantitative analysis
• Consultant Histopathologist support
• Image analysis
• Vectra Polaris image scans allows digital analysis to integrate seven-color multispectral imaging with whole-slide scanning
• Aperio Digital image scans allow digital analysis of full face and TMA slide sections
• ImageQuant – segmentation and TMA algorithms