IMMUNOHISTOCHEMICAL DETECTION OF THROMBOSPONDIN-1 IN FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE

Thrombospondin-l (TSP) is a 450-KD glycoprotein that was initially dis covered in the platelet a-granule. It now appears that TSP is intimate ly involved in the regulation of a variety of cellular functions and c ell-to-cell interactions, Recently, it has been demonstrated that TSP functions as a p53-dependent inhibitor of angiogenesis in cultured fib roblasts from Li-Fraumeni patients and therefore may be an important f actor involved with tumor invasion and metastasis. It has previously b een demonstrated that TSP can be detected in frozen tissue sections by immunohistochemical methods, Our objective in this study was to deter mine the optimal antigen retrieval (AR) protocol for detection of TSP in formalin-fixed, paraffin-embedded tissue by using tissue sections f rom patients with invasive transitional cell carcinoma of the bladder. The optimal AR protocol was determined utilizing a variety of heating conditions and antigen retrieval buffers. Our results demonstrate tha t TSP can be reliably detected in paraffin-embedded tissue by immunohi stochemical techniques that utilize AR with high-temperature microwave heating and a low-pH Tris-HCl buffer. The importance of this method i s that it allows the reliable detection of TSP in archival tissue. Thi s should facilitate further investigation into TSP's role in the regul ation of cellular processes, including its influence on tumor angiogen esis and metastasis.