Ultravision one hrp polymer kit

The IHC was done as previously described by us^{55 (link)}. Tumors collected from in vivo study were dehydrated, embedded in paraffin and sectioned into 5–10 micron sections using microtome. The sections were gently placed on positively charged slides, deparaffinized and rehydrated using xylene, ethanol and double-distilled water. The sections were further boiled in 10 mM sodium citrate buffer (pH 6.0) for antigen unmasking and incubated in 3% hydrogen peroxide solution. After blocking with 5% goat serum, sections were incubated overnight with primary antibodies for HER2, β-catenin and cleaved PARP. Next day, using Ultravision ONE HRP polymer kit (Thermofisher scientific, Fremont, CA), the slides were stained as per the manufacturer’s instructions. Further, these sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted using Permount and then imaged using Olympus microscope (Olympus America Inc, Center Valley, PA).

The immunohistochemistry (IHC) was performed as previously described by us28 (link)29 (link). Briefly, fixed tumor tissues were dehydrated and embedded in paraffin. Tissues were sectioned into 5 μm thick sections using microtome (Leica Microsystems Inc., Buffalo Grove, IL). The sections were deparaffinized as well as rehydrated using xylene, ethanol and double-distilled water washes. Unmasking of antigen was done by boiling the sections in 10 mM sodium citrate buffer (pH 6.0). Sections were washed and incubated in 3% hydrogen peroxide solution. The sections were blocked with 5% goat serum and incubated with primary Antibodies for cleaved caspase 3 (1:300), LC3B (1:1000) and p62 (1:100) overnight at 4 °C. Next day the slides were stained using Ultravision ONE HRP polymer kit as per the manufacturer’s instructions (Thermofisher scientific, Fremont, CA). Counterstaining of sections was performed with Mayer’s hematoxylin and dehydrated. The slides were mounted using Permount (Fisher scientific, Fair Lawn, NJ) and imaged using Olympus microscope (Olympus America Inc, Center Valley, PA). Antibodies for cleaved caspase 3 (Cat.#9661S), LC3B (Cat.#3868S) and p62 (Cat.#5114S) were purchased from Cell Signaling. TUNEL assay was carried out according to manufacturer’s protocol (TUNEL assay kit was purchased from Calbiochem, San Diego, CA, USA).