Six-micrometre thick serial sections of formalin-fixed, paraffin-embedded OSC and NDOLP tissue were deparaf-finized by the xylene-ethanol sequence. For antigen retrieval, the sections were microwaved at 500 W for 10 min., after which endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution. Adjacent sections were incubated with monoclonal antibodies anti-TP (P-GF.44C Neo-Markers, Fremont, CA) diluted 1:100 for 1 hr at room temperature; anti-CD34 (QB-END 10; Bio-Optica, Milan, Italy) pan-endothelial marker diluted 1:50 for 1 hr at room temperature; anti CD68 (KP1; Dako) macrophage marker diluted 1:100 for 1 hr at room temperature [22, 28 (link)]. The bound antibody was visualized using biotinylated secondary antibody, avidinbiotin peroxidase complex, and 3-amino-9-ethylcarbazole or 3,3 diaminoben-zidine. Nuclear counterstaining was performed with Gill's haematoxylin number 2 (Polysciences, Warrington, PA) [18 ]. Primary antibody was omitted in negative controls.
TP expression was determined in five 400 × fields by the image analysis system (Quantimet 500 Leica) and TP positivity was evaluated on the basis of stained epithelial, macrophages and endothelial cells in terms of MVD [18, 24, 29 ]. Endothelial cells were identified as CD34- and TP-positive cells and MVD was evaluated in terms of both TP and CD34 immunostained vessels accordingly to Weidner's method with slight modifications [29 (link)]. Macrophages were identified as CD68- and TP-positive cells. Mean values ± standard deviation of epithelial cells, macrophages and MVD in both NDOLP and OSC was determined for each section and group of samples. Median value of epithelial cells, macrophages and MVD in OSC positive to TP was determined for each section and group of sample and was used as a cut-off to distinguish between high and low TP reactivity.
TP expression was determined in five 400 × fields by the image analysis system (Quantimet 500 Leica) and TP positivity was evaluated on the basis of stained epithelial, macrophages and endothelial cells in terms of MVD [18, 24, 29 ]. Endothelial cells were identified as CD34- and TP-positive cells and MVD was evaluated in terms of both TP and CD34 immunostained vessels accordingly to Weidner's method with slight modifications [29 (link)]. Macrophages were identified as CD68- and TP-positive cells. Mean values ± standard deviation of epithelial cells, macrophages and MVD in both NDOLP and OSC was determined for each section and group of samples. Median value of epithelial cells, macrophages and MVD in OSC positive to TP was determined for each section and group of sample and was used as a cut-off to distinguish between high and low TP reactivity.
