Immunohistochemical Analysis of Bone Metastasis

The bones were fixed in 10% neutral buffered formalin, decalcified in Mielodec (Bio-Optica, Milan, Italy) and embedded in paraffin; serial sections (4 μm) for each specimen from three mice per group were stained with hematoxylin and eosin to verify bone metastasis presence. After antigen retrieval (95 °C for 20 min at pH 6 in antigen-unmasking solution, Vector Laboratories, Burlingame, CA, USA), sections were treated for 10 min with 0.1% (v/v) H₂O₂ and blocked with normal serum. Immunostaining was performed on bone specimen slices from three mice with anti-Twist (1:200), anti-Snail (1 µg/mL), anti-human HGF (3 μg/mL) or anti-mouse HGF (10 μg/mL) antibody, using a streptavidin-biotin system (ABC kit, Santa-Cruz Biotechnology) and diaminobenzidine substrate, and counterstaining with Meyer’s haematoxylin [25 (link)]. Negative-control sections were subjected to the same staining procedure without the primary antibody.