Immunohistochemistry Protocol for Phospho-Protein Analysis

Samples were fixed in 10% formalin and embedded in paraffin. After deparaffinization, slides were washed with 9.83% NaCl for 3 min followed by a PBS wash and a wash in distilled water for 5 min. Heat induced antigen retrieval was performed with pressure cooker (2100 Retriever, PickCell Laboratories B.V.) and R-Buffer A (pH6.0, Electron Microscopy Sciences). Sections were incubated with 2% H₂O₂ in Methanol for 15 minutes for endogenous peroxidase quenching and washed and blocked for non-specific binding in 1% BSA in PBS-Triton 0.3% v/v (PBST) for 1 hour. Subsequently, sections were sequentially incubated with primary antibody at 1:100 dilution for 1 hour, secondary peroxidase goat anti-rabbit IgG antibody (Vector) at 1:250 dilution for 1 hour and Tyramide Signal Amplification (TSA) Fluorescein System (Perkin Elmer, Cat.: NEL701A) according to kit manual. Slides were mounted with Vectashield mounting medium with DAPI (Vector), photographed with Nikon C2 Confocal Microscope system and subsequently stained with Hematoxylin and Eosin. p-AKT (Thr308) p-ERK (Thr202/Tyr204), p-S6 (Ser235/236), and CC3 (Asp 175) were obtained from Cell Signaling, Inc. Ki67 staining was performed as previously described (19 (link)). Quantification of Ki67 staining was performed by scoring the nuclei of cells from each lesion type found in a minimum of ten high-powered fields.