Immunohistochemistry (IHC) Core: What we Offer
The principal purpose of the IHC Core is the development and validation of immunohistochemical methods for investigators. This is accomplished under the experimental pathology umbrella; in collaboration with the BRC and the histopathology core by providing the following services:
- Basic histopathologic services and guidance for specimens bound for histochemistry, immunohistochemistry and immunoflorescence
- Extensive options for special and routine histochemical and immunohistochemical labeling of both human and animal tissues in fresh, frozen and paraffin embedded media including 2 color (double stain) chromogenic detection
- Manual immunohistochemical labeling with a variety of detection systems including, tyramide and polymer detection systems
- Automated, high throughput immunohistochemistry using two, 20 slide capacity Ventana Medical System NEXes modules and two, 20 slide Discovery modules
- A catalog of over 1,000 antibodies against a wide variety of targets in both human and animal tissues
- Consultation and guidance in planning histopathology and IHC related experiments as well as informal training and support for all investigators
Antibody Validation & Verification
In order to validate antibodies the IHC core laboratory uses tissue microarrays (TMA) constructed from animal and human frozen or archival tissues. In collaboration with the Biorepository Center (human) and the histopathology core (animal), control TMAs are prepared for use by the IHC core for antibody validation and verification (+/- controls). Investigators may also have TMAs of their sample sets prepared for use in collecting IHC and other histopathology data. For more information, please visit the BRC website.
TMAs are used for testing in order to maximize the efficien.
cy of the validation process. Antibody performance is assessed in relevant human and mouse normal and tumor tissue arrays to demonstrate antibody performance over a broad spectrum of tissue types (including negative samples). In addition, TMAs reduce the need to screen tissues for either general or specific marker immunoreactivity for use as positive and negative controls. This ensures reproducibility and enables quality control of antibody performance throughout the project lifecycle. The core lab uses a standardized approach to validate antibodies. Tissue samples are tested under a variety of conditions and treatments with the goal of producing the best possible signal to noise performance for an antibody. Validation procedures for cells, fresh/frozen and FFPE tissues can include but are not limited to:
- Staining on fresh smears, touch-preps cytospins and frozen tissues when appropriate
- Cell culture material of known target expression levels prepared (smears, cytospins, frozen and FFPE) and used to verify target specificity
A variety of antigen retrieval techniques including:
- Different buffers for heat induced antigen retrieval (HEIR) with microwave, pressure cooker and steamer systems
- Different proteolytic enzyme digestion (alkaline endopeptidase, trypsin, proteinase K + others)
- A variety of blocking agents used to reduce non-specific staining and endogenous background
- Sample material subjected to phosphatase treatment to verify target phospho-specificity
- Competition assay can be performed to demonstrate specificity
- Primary and secondary antibody serial dilutions to determine dynamic range and end point
- Different detection methodologies (SAB, polymer, multimer and tryamide amplification systems and reporter systems (peroxidase and alkaline phosphatase)
- Antibody biotinylation (ARK) for same species antibodies
- New lot testing (verification) to ensure reproducibility
In collaboration with the Histology core, whole slide scanned (Leica SCN 400F Whole Slide Scanner) images of antibody validation process are collected for documentation and shared with PIs and their staff. Antibody validation scanning is provided "free of charge" as part of the validation procedure in order to facilitate joint evaluation of antibody performance. Once antibody conditions and performance is established, sample set staining can commence. Sample set scanning is performed by the Histology core lab as a service. For more information, please contact Lisa Mara of the histopathology core lab.
If initial validation testing does not yield suitable results, additional testing may be required and a new antibody may need to be obtained . Currently, the average number of tests required to validate an antibody is approximately 9 slides (subject to change and target species dependent). Antibodies that have not been routinely used by the laboratory within the previous 3 months are subject to verification, including new antibody lots. Antibodies are verified by testing the last known set of validated conditions with the same TMA that was used during validation (if available). Once verified, investigator samples sets will be run with the pre-established antibody conditions without further validation. If antibody performance cannot be reproduced from previously established conditions, the antibody may undergo revalidation or an alternate antibody may be chosen (including searches for alternate commercial antibodies not in the core catalog). The core will discuss these options plus exploring other possible approaches with PIs and their staff.