All immunostained slides were evaluated twice by experienced pathologists (LJH and LKH) blinded to the clinical details. IHC was performed as described previously [43 (link)]. Tissue sections were immunostained with specific antibodies against Cav-1 (1:800, BD Biosciences, Franklin Lakes, NJ, USA) and SNAIL (1:100, Santa Cruz Biotechnology Inc., Dallas, TX, USA) using a Bond-Max autostainer system (Leica Microsystems, Buffalo Grove, IL, USA). Negative controls were processed in the absence of primary antibodies. The Cav-1 antibody stained the cytoplasmic borders and cytoplasm of cancer cells. The SNAIL antibody mainly stained the cytoplasm of cancer cells. The staining intensity in cancer cells was initially graded according to the following criteria: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. Samples were grouped according to staining intensities of 0–1 (low expression) and 2–3 (high expression). These samples were also grouped according to staining intensity as described above (Fig. 1b).
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