Immunohistochemical detection of progranulin (PGRN/GP88/GEP) in tumor tissues as a cancer prognostic biomarker

Abstract

Immunohistochemistry is a well characterized and robust staining technique for the identification and localization of proteins in biological tissues. The following methodology describes the process for identifying progranulin (PGRN/GP88/GEP) in the cellular cytoplasm of target tissues such as tumor biopsies. Such tissue is collected from cancer patients and stabilized using formalin-fixed paraffin embedding after which 4–5 μm sections of the tissue are mounted on positively charged microscope slides. After deparaffinization using ethanol, the tissue is processed through an antigen retrieval process using citric acid at pH 6.4. A monoclonal antibody (Clone AG03) against human PGRN/GP88/GEP is then contacted with the tissue before a horseradish peroxidase (HRP) conjugated antibody is employed to detect AG03 bound to PGRN/GP88/GEP. Subsequent application of the HRP substrate results in color generation. Using a light microscope, a trained user can “score” the percentage of cells stained and intensity of such staining. Scoring ranges from 0 (no staining) to 3+ (strong cytoplasmic staining in >10% tumor cells). When scoring is correlated to patient outcome, we have demonstrated that a patient with tumor PGRN/GP88/GEP staining of “3+” has a fivefold increased risk of disease recurrence within 5 years.