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For the simultaneous detection of CD34 and laminin in melanoma tissue, slides were incubated with monoclonal mouse anti-human CD34 (QBend10; Dako) at a dilution of 1:40 for 30 minutes and placed in protein-blocking solution (Dako) for 10 minutes. Antibody binding was detected with Alexa Fluor 488 conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) for 30 minutes at a dilution titer of 1:400. After 10 minutes of protein blocking, slides wereincubated with polyclonal rabbit anti-laminin antibody (Sigma-Aldrich, St Louis) at a dilution titer of 1:200 for 30 minutes at room temperature. After protein blocking for 10 minutes,antibody binding was detected with Alexa Flour 594-conjugated goat anti-rabbit IgG (Invitrogen) for 30 minutes at a dilution titer of 1:400. Slides were rinsed in buffer and mounted in aqueous mounting medium (Faramount; Dako). For all staining procedures, secondary antibody was omitted in negative control experiments. For the reconstruction of blood vessels and VM patterns, we examined 7 serial sections from each of 4 areas of highly vascularized metastatic uveal melanoma to the liver co-localizing to VM patterns. ¼±Çó·ÒëÕâ¶ÎÓ¢ÎÄ£¬Ð»Ð»£¬Íò·Ö¸Ðл£¡£¡£¡ |
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