Immunostaining of Tumor Tissue Sections

Hematoxylin and eosin staining was performed with paraffin sections by Duke Pathology Laboratory (Durham, NC, USA). Immunostaining of OCT-embedded frozen subcutaneous tumor tissue sections were performed as previously described.^{44 (link)} Five-micrometer-thick sections were fixed in 100% methanol for 15 min, blocked with 10% horse serum in PBS (Invitrogen) for 1 h and then incubated with antibodies against Ki-67 (SP6) (Thermo Fisher Scientific, Grand Island, NY, USA); cleaved caspase 3 (#9661), N-cadherin (#13116), E-cadherin (#3195) and β1-Integrin (#9699) (Cell signaling Technology) for 1 h at room temperature or 4 °C overnight. Sections were then washed five times in PBS with 0.05% Tween-20, followed by detection with Alex 555-conjugated secondary antibody (Thermo Fisher Scientific) and counterstaining with 1 μg/ml Hoechst 33258 for 1 min. For immunostaining, paraffin-embedded lung tissue sections were dewaxed in 100% xylene for 5 min followed by sequential 5-min treatments of 100, 90 and 70% ethanol, antigen unmasking by boiling in 10 mM citrate buffer for 10 min and then undergo immunostaining as described above with a primary antibody against Melan-A (HPA048662) (Sigma) and detection with a Dylight 549-conjugated secondary antibody. Fluorescent pictures were taken and processed using the Olympus BX41 microscopic imaging system (Olympus, Center Valley, PA, USA).