What they do – The HER-2/ER/PR Reference IHControls™ are on-slide immunohistochemistry (IHC) staining controls. Positive controls are important for testing if the immunohistochemical assay worked properly. HER-2/ER/PR formalin-fixed changes in stain intensity (beyond the normal variability) likely indicate a problem. Corrective action may be required, at the discretion of the laboratory. Changes in stain intensity of the IHControls™ can be due to variability with antigen retrieval, reagents, protocols, or the instrument.
What they’re made of – Our controls are made of 8-micron diameter glass microbeads coated with a portion of the antigen – – HER-2, ER, or PR (see figure below). The microbeads are dispersed within a liquid matrix that solidifies after dispensing onto a microscope slide. The solidified matrix is porous to IHC staining reagents. Because the matrix is porous, antibodies and other reagents that are applied during the IHC staining procedure will stain the microbeads in a similar fashion as they stain tissue sections.
Figure legend: IHControls™ are made up of a suspension of hundreds of thousands of small glass microbeads (per vial), each microbead being the approximate size of a cell. The microbeads are coated with antigen fragments (peptides) that are immunoreactive with one or more of the antibodies commonly used in immunohistochemistry.
IHControls™ behave like formalin-fixed, paraffin-embedded tissue controls – The IHControls™ are already formalin-fixed. Without antigen retrieval, staining is typically weak or negative. In tissue sections, formalin fixation causes cross-links within a single protein or between adjacent proteins. If a protein cross-link brings another protein at or near the antibody epitope, then it can sterically interfere with antibody binding. Antigen retrieval reverses formaldehyde-induced crosslinks and dissociates proteins that are sterically interfering with antibody binding. (1) We use the same formaldehyde fixation chemical reaction on the IHControls™. During manufacture, we incubate peptide-coated glass microbeads with formaldehyde and an irrelevant protein (casein). This causes the irrelevant protein to be covalently bound to the peptide, blocking antibody binding to the peptide epitope. Antigen retrieval reverses the reaction and re-exposes the peptide epitope for binding to the antibody.
1. SA Bogen, K Vani, & SR Sompuram. 2009. Molecular Mechanisms of Antigen Retrieval: Antigen Retrieval Reverses Steric Interference Caused By Formalin-Induced Crosslinks. Biotechnic & Histochem. 84(5):207-215.