Mach 3 rabbit hrp

Human tissue arrays were obtained from Folio (Catalogue number: FOLIO-800). Deparaffinization of tissue slides was performed at 60°C for 1 hr. After 10 min of cooling, tissues were hydrated in two separate baths of xylene for 5 minute each, followed by a bath in 2% hydrogen peroxide/methanol solution for 30 minutes and finally incubated in 3 different concentrations of ethanol solutions (100%, 95%, 70%). After neutralizing in water, antigen retrieval was performed in citrate buffer (pH 6) at 100–120°C for 10 minutes. Sections were cooled at 4°C for 30 min and subsequently blocked using 5% BSA/Tris buffer solution/ 0.1% Tween (TBST) for 30 min at room temperature. Blocked tissues were incubated with unconjugated primary antibody overnight: anti-EGFR (E746-A750del Specific) or anti-PKCα (Cell Signaling), both diluted 1:50 in block solution. Tissues were washed for 5 min 3x with TBST and incubated with conjugated secondary antibody (Biocare MACH 3 Rabbit HRP). Sections were then washed and counterstained with nuclear stain and mounted in Vectashield mounting medium (Vector Labs). Microscopy was performed using a Zeiss microscope. Optical density (OD) for PKCα staining were calculated by ImageJ software version 2 (https://imagej.nih.gov/ij/).