Hypoxic regions in mouse tissues were visualized using the Hydroxyprobe-1 kit (Chemicon International, Temecula, CA, USA). These experiments were approved by the animal experimentation ethics committee, according to local and governmental regulations.
Briefly, mice were injected intravenously with pimonidazole (60-mg/kg body weight), which forms adducts from reductively activated pimonidazole in hypoxic cells. Mice were killed 90 min later by asphyxiation in CO2. The Hydroxyprobe-1 monoclonal antibody was biotinylated using D-biotinyl-ɛ-aminocaproic acid N-hydroxysuccinimide ester (Boehringer Mannheim) and was used to stain pimonidazole adducts in formalin-fixed paraffin-embedded tissues according to recommendations by the manufacturer.
Briefly, mice were injected intravenously with pimonidazole (60-mg/kg body weight), which forms adducts from reductively activated pimonidazole in hypoxic cells. Mice were killed 90 min later by asphyxiation in CO2. The Hydroxyprobe-1 monoclonal antibody was biotinylated using D-biotinyl-ɛ-aminocaproic acid N-hydroxysuccinimide ester (Boehringer Mannheim) and was used to stain pimonidazole adducts in formalin-fixed paraffin-embedded tissues according to recommendations by the manufacturer.
