Specimens from primary tumor or metastases were obtained from patients undergoing surgery within four weeks of the PET/CT. The specimens were placed in formalin and paraffin embedded within 24 h. The samples were cut in sections of 4 µM and dewaxed through xylene to tap water. For antigen retrieval the sections were treated with proteinase K for 5 min. This was followed by a blocking step with Peroxidase-Blocking Solution (Agilent, S2023) and pre-incubation in 2% BSA for 10 min. For visualizing the intensity and distribution of integrin, αvβ3 sections were incubated with primary antibody (Absolute Antibodies, Ab00890-23.0) in a 1:50 dilution in 2% BSA overnight at 40 °C [19 (link)].
For visualization, the sections were incubated with Envision+ system Anti-Rabbit (Agilent, K4003) for 45 min followed by incubation with DAB+ system (Agilent, K3468) for 10 min. Counterstaining was performed with Mayer’s Hematoxylin. The sections were visually evaluated regarding αvβ3 intensity.
For visualization, the sections were incubated with Envision+ system Anti-Rabbit (Agilent, K4003) for 45 min followed by incubation with DAB+ system (Agilent, K3468) for 10 min. Counterstaining was performed with Mayer’s Hematoxylin. The sections were visually evaluated regarding αvβ3 intensity.
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