Anti gli2

The immunohistochemistry (IHC) was performed as previously described by us [24 ]. Briefly, paraffin-embedded tissues were sectioned into 5μm thick sections using microtome (Leica Microsystems Inc., Buffalo Grove, IL). After deparaffinization and rehydration, antigens were retrieved by boiling the sections in 10 mM sodium citrate buffer (pH 6.0). The slides were washed with distilled water and incubated in 3% hydrogen peroxide methanol solution. The sections were then washed, blocked in 200 μl of blocking solution (5% goat serum diluted) and incubated with anti–HER2 (1:150) (Abcam, Cambridge, MA) or anti-GLI2 (1:50) (Cell signaling, Danvers, MA) overnight at 4°C. Next day primary antibody was removed and the sections were washed with wash buffer followed by 30 minute incubation with Ultravision ONE HRP polymer (Thermofisher scientific, Rockland, IL) as per the manufacturer’s instructions. Subsequently, sections were washed with wash buffer and incubated with DAB Plus chromogen for 15–20 minutes. The sections were counterstained with hematoxylin and dehydrated. The slides were mounted using Permount (Thermofisher scientific, Rockland, IL) and analyzed under a bright field Olympus microscope (Olympus America Inc).