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Paraffin-embedded tissue blocks from formalin-fixed tumor samples were sectioned, dewaxed, and rehydrated using standard techniques. For immunofluores-cent staining, 7- μm cryo-sections were fixed in acetone for 10 min and dried in air for 1 h. Following blocking of nonspecific binding with 5% filtered goat serum in PBS for 1 h, sections were incubated with primary antibody for 1 h at room temperature. After a PBS wash, sections were incubated with secondary antibody Alexa-Fluor 594 goat anti-rabbit, Alexa-Fluor 555 goat anti-rat (0.5 A g/mL, Molecular Probe, Invitrogen), rabbit anti-rat FITC (1:20, DAKO) or Texas Red–conjugated streptavidin (1:100, Molecular Probe, Invitrogen) for 30 to 60 min in the dark. Following a PBS wash, slides were mounted in DAKO Fluorescent Mounting Medium. Vascular density and vessel size of tumors were quantified after staining ECs with rat anti-mouse CD31/PECAM (2 μg/mL, Clone ER-MP12, Acris GmBH). Fluorescent microscopy images were acquired at a resolution of 1,300 1,030 pixels. [ Last edited by yin198716 on 2013-4-30 at 18:33 ] |
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