Immunohistochemistry (IHC) is a technique that uses antibodies to allow investigators to visualize the expression levels and distribution of specific proteins (antigens) in cells and tissues. Due to the variability among antibodies and staining techniques, IHC requires significant knowledge of basic histology, available immunological reagents, antigen retrieval techniques and immunohistochemical procedures. Skilled technologists in the Core provide customized IHC using the automated Ventana Discovery Ultra platform or by manually staining the samples on the benchtop.
Both Fluorescent (FISH) and chromogenic (ISH) in situ hybridization is available using the RNAscope platform from Advanced Cell Diagnostics. This technique uses novel probes for detection of target RNA within intact cells and tissues.
Investigators are encouraged to contact the Core before starting a project or buying antibodies. Our skilled technologists will offer careful advice about staining strategies and help determine the best antibodies and positive control tissues for a successful result.
Antibody Titration and Optimization
Determination of the proper conditions such as antigen retrieval protocol and antibody concentrations for optimal staining.
Staining of frozen or paraffin-embedded tissue sections with chromogenic (horseradish peroxidase-DAB, alkaline phosphatase) or fluorescent-labeled antibodies. Single, double and triple staining are available. For labelling with more three antibodies see “Multiplex IHC” below.
Staining of paraffin-embedded tissue sections with up to 7 antibodies using Akoya OPAL technology. This method utilizes individual Tyramide Signal Amplification (TSA)-conjugated fluorophores to detect multiple targets on a tissue section. Primary antibodies made in the same species can be used without species cross-reactivity. The Core is developing antibody panels similar to those used for Flow Cytometry.
In Situ Hybridization (F/ISH)
To investigate RNA expression in intact tissue the Core provides the RNAscope platform in both chromogenic (blue/brown/red) and fluorescent modes. The probe design strategy is similar to fluorescence resonance energy transfer (FRET), in which two independent probes (double Z probes) have to hybridize to the target sequence in tandem in order for signal amplification to occur. Since it is highly unlikely that two independent probes will hybridize to a non-specific target right next to each other, the design ensures selective amplification of target-specific signals. Visit the ACD website for more information.
The Immunohistochemistry section of the Imaging Core is staffed by two senior technologists, each with many years’ of experience processing and staining tissues for research and pathology. They offer a full complement of tissue cutting and staining procedures. Automated immunostaining and in-situ hybridization (F/ISH) is done on the Ventana Discovery Ultra platform that generates very reproducible results. Manual IHC and F/ISH can be done on the benchtop for individual applications. We will be happy to provide assistance with the choice of antibodies, staining workups and evaluation. Stained slides can be imaged using a variety of imaging systems in the Core. Expert assistance is available for downstream image analysis
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