A series of formalin-fixed and paraffin-embedded tissue samples obtained from 86 cases of CMCTs were utilized. Histological diagnosis was performed on almost six slides for each tumour sample stained with haematoxylin-eosin and Undritz method (Merck, Darmstadt, Germany), specific for red-blue methachromatical MCs identification [22 (link)]. Accordingly to Patnaik et al. [35 (link)], the cases were classified as follows: 31 were G1, corresponding to well differentiated CMTC, 27 were G2, corresponding to intermediate differentiated CMTC, and 28 were G3, corresponding to poorly differentiated CMTC.
For the evaluation of MVD and VEGF expression, a three-layer biotin-avidin-peroxidase system, as previously described was adopted [22, 36 (link)]. Briefly, six serial sections, for each tissue samples, were cut. After heating, slides were incubated with the rabbit polyclonal antibody anti-factor VIII-related antigen (FVIII-RA) (Dako, Glostrup, Denmark), used as an endothelial marker and with the rabbit polyclonal anti-VEGF (Santa Cruz Biotechnology, CA, USA) antibody. The bound antibodies were visualized by using biotinylated secondary antibody, avidin-biotin peroxidase complex and 3-amino-9-ethylcarbazole or fast red (Dako). Nuclear counter-stained was performed, for each tissue sample, with Gill's haematoxylin (Polysciences, Warrington, PA, USA). A double stain was also performed by using anti-FVIII-RA antibody and Undritz method to mark on the same slide both endothelial cells and MCs [22 (link)]. As a negative immunohistochemical control, no primary antibody was added.
The slides were morphometrically evaluated by using an image analysis system (Quantimet 500 Leica Microsystems, Wetzlar, Germany). Ten most vascularized areas (‘hot spot’) were selected at low magnification and single red-brown stained endothelial cells, endothelial cell clusters and microvessels, clearly separated from adjacent microvessels, tumour cells and other connective tissue elements and MC we counted at x400 fields and x1000 fields in oils [1, 20, 22 (link)]. Finally, serial sections were also evaluated for MCs reactive to VEGF.
For the evaluation of MVD and VEGF expression, a three-layer biotin-avidin-peroxidase system, as previously described was adopted [22, 36 (link)]. Briefly, six serial sections, for each tissue samples, were cut. After heating, slides were incubated with the rabbit polyclonal antibody anti-factor VIII-related antigen (FVIII-RA) (Dako, Glostrup, Denmark), used as an endothelial marker and with the rabbit polyclonal anti-VEGF (Santa Cruz Biotechnology, CA, USA) antibody. The bound antibodies were visualized by using biotinylated secondary antibody, avidin-biotin peroxidase complex and 3-amino-9-ethylcarbazole or fast red (Dako). Nuclear counter-stained was performed, for each tissue sample, with Gill's haematoxylin (Polysciences, Warrington, PA, USA). A double stain was also performed by using anti-FVIII-RA antibody and Undritz method to mark on the same slide both endothelial cells and MCs [22 (link)]. As a negative immunohistochemical control, no primary antibody was added.
The slides were morphometrically evaluated by using an image analysis system (Quantimet 500 Leica Microsystems, Wetzlar, Germany). Ten most vascularized areas (‘hot spot’) were selected at low magnification and single red-brown stained endothelial cells, endothelial cell clusters and microvessels, clearly separated from adjacent microvessels, tumour cells and other connective tissue elements and MC we counted at x400 fields and x1000 fields in oils [1, 20, 22 (link)]. Finally, serial sections were also evaluated for MCs reactive to VEGF.
