Lymphatic vessels identified with podoplanin. Comparison of immunostaining with three different detection systems

The aim of the study was to find the optimal immunostaining protocol for monoclonal mouse anti human podoplanin antibody clone 18H5, which is less studied. We tested three detection systems for different antibody dilutions and antigen retrieval methods applied on normal and tumor breast tissues, lip squamous cell carcinoma, kidney tumors and testis carcinoma. The interpretation was linked to the background staining, specificity for the lymphatic endothelium and cross-reactivity. The ABC method gave a high background even the antibody dilution was increased. No immunostaining was obtained with Envision detection system for any dilution or type of antigen retrieval. We obtained the best results using LSAB+ working system for an antibody dilution of 1:500 with no background and podoplanin expression was restricted to the lymphatic endothelium. We consider that the standardization of immunostaining protocol is the first step for an optimal interpretation of the podoplanin expression using less studied antibody clones.