Immunohistochemical Detection of Glypican-5 in Paraffin-em bedded Material: An Optimized Method for a Novel Research Antibody

摘要

Glypican-5 (GPC5) is a cell surface heparan sulfate proteoglycan and 1 of 6 closely related members of the glypican family in mammals. Glypicans are predominantly expressed during development in cell-specific and tissue-specific contexts, and the expression of some is linked to developmental disorders and several visceral malignancies. We have previously shown that the region of amplification at 13q31.3 in a subset of rhabdomyo-sarcomas contains the GPC5 locus, and by copy number and gene expression analyses, that GPC5 is consistently expressed and upregulated in amplified tumors. As the immunohistochemical profile of GPC5 is untested, our aim was to optimize a commercially available anti-human GPC5 antibody for immunohistochemical use in formalin-fixed and paraffin-embedded (FFPE) tissue. Quantitative real-time polymerase chain reaction analyses of normal tissue samples indicated that the brain and testis highly expressed GPC5. High protein expression in these tissues and a cell line constructed to overexpress GPC5 were demonstrated by Western blotting. These normal tissues and the isogenic cell line were FFPE, and immunohistochemical expression of GPC5 was assessed using different methods of antigen retrieval, detection, and primary antibody concentration. The optimum conditions for detection were by heat-induced antigen retrieval, in sodium citrate buffer at pH 6. Enzyme-mediated retrieval did not produce effective detection, producing weaker, less well-localized GPC5 expression. We demonstrate that anti-human GPC5 antibody is amenable to use in FFPE tissue and with the optimized protocol we describe shows specific cellular localization and good staining intensity with minimal background staining.