Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is one of the most widely-used diagnostic procedures in modern medicine. This method is used to determine the expression levels and subcellular localisations of specific proteins in a tissue sample.

We seek to gain further knowledge about the relationship between protein expression and the development of cancer, with an aim to increase our mechanistic insight and identify possible prognostic markers for cancer patients. We are currently conducting IHC studies of a panel of selected proteins involved in relevant processes such as cell cycle regulation. We have identified a large number of potential cancer-related proteins in sections from patients with prostate and colorectal carcinomas. The relationship between the protein expression and clinicopathological variables including survival for the patients will be examined, with the main goal of identifying potential prognostic biomarkers for patients with these cancer types.

Principles of ImmunohistostainingPrinciples of Immunohistostaining1) Tumor tissue containing the protein or antigen one seeks to target is selected. 2) A primary antibody is conjugated to a reagent, such as an enzyme that can catalyze a color producing reaction or a fluorescent tag, binds to the antigen. 3) To amplify the signal, the indirect visualization system uses a conjugated secondary antibody, which binds to an unlabeled primary antibody. 4)The slide is dipped in a substrate bath. 5) The slide is dipped in a bath containing a counterstain. 6)The enzyme reacts with 3, 3’-Diaminobenzidine to produce a brown staining when the targeted antigen is present. 7) Example where stained cells and structures appear brown.

Method

In IHC, we use antibodies that recognise and bind to a specific region within the protein of interest. Different methods can be used for visualisation of this bound antibody. In the direct method, the antibody is conjugated to a reagent, such as an enzyme, that catalyses a colour producing reaction or a fluorescent tag. The indirect visualization system uses an enzyme-conjugated secondary antibody, which binds to an unlabelled primary, to amplify the signal, The enzyme reacts with a chromogen substrate such as 3, 3’-Diaminobenzidine (DAB) or 3-Amino-9-ethylcarbazole (AEC), producing a brown or red end product, which visualizes where the targeted protein is present. The chromogenic signal can be amplified using different methods. We use the polymer-based detection method, where antibodies and enzymes are conjugated to a polymer backbone, enhancing the sensitivity and specificity of the signal.

Membraneous FlotillinsMembranous Flotillin 2 expression in mammae carcinomas. Panel A shows low total score Flotillin 2 expression, whereas Panel B represents intermediate Flotillin 2 expression and Panel C high total score Flotillin 2 expression.

Seeking Prognostic Biomarkers

We are currently using AutostainerLink 48 (Dako, Agilent technologies) for our IHC projects. We aim at identifying a large number of potential cancer related proteins in sections from patients with prostate and colorectal carcinomas. The relation between the protein expression and clinicopathological variables including survival for the patients will be examined, with a main goal of identifying potential prognostic bio­markers for patients with these cancer types.