LAUSR

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.