Plus kit hp2

Vessel perfusion and hypoxia studies were performed according to the protocol previously described in [27 (link)]. Mice bearing tumors (vehicle- or drug-treated) were intravenously injected with 100 mg of biotinylated tomato lectin (derived from Lycopersicon esculentum) (Vector Labs, Newark, CA, USA, #B-1175) prepared in 100 μL of 0.9% NaCl, followed by intraperitoneal injection with 60 mg/kg of pimonidazole hydrochloride. The tumors were harvested 2 h after the lectin perfusion and hypoxia injection, fixed in 10% formalin, and processed for subsequent experiments. To visualize the productive microvessels, IHC was performed using the streptavidin–biotin peroxidase complex method according to the manufacturer’s instructions (Lab Vision Corporation, Fremont, CA, USA). To determine the extent of the hypoxia, the hypoxic regions within the tumors were identified by staining the sections with the Hypoxyprobe Plus Kit HP2 according to the manufacturer’s instructions (Hypoxyprobe Inc., Burlington, MA, USA). For the quantification analysis, 10 random 0.159 mm² fields were captured at a magnification of 100× and counted on each IHC-stained slide.

Each mouse bearing tumor xenografts (vehicle- or drug-treated) was intravenously injected with 100 mg of biotinylated Lycopersicon Esculentum (Tomato) Lectin (VectorLabs #B-1175) prepared in 100 μL of 0.9% NaCl, as previously described [23 (link)]. The tumors were harvested 10 min after lectin perfusion and fixed in 10% formalin for paraffin embedding before obtaining 5-μm sections.
To determine the extent of hypoxia in tumor tissues, mice bearing tumors (vehicle- and drug-treated) were intraperitoneally injected with 60 mg/kg pimonidazole hydrochloride 1 h before tumor harvest.
To visualize productive microvessels, immunohistochemistry was performed using the streptavidin-biotin peroxidase complex method, according to the manufacturer’s instructions (Lab Vision Corporation, Fremont, CA, USA). Hypoxic regions of tumors were identified by staining the sections with Hypoxyprobe plus Kit HP2 according to the manufacturer’s instructions (HypoxyProbe Inc., Burlington, MA, USA).