BRAF V600E mutation–specific immunohistochemistry

Immunohistochemistry was performed on formalin-fixed, paraffin-embedded (FFPE) tissue using a mutation-specific antibody to BRAF V600E protein (VE1 clone; ready-to-use dilution; Ventana Medical System, Tucson, AZ) in a Leica Bond-Max automated immunostainer (Leica Biosystems, Deer Park, IL). Three-μm thick sections mounted on adhesive glass slides were deparaffinized and subjected to heat-induced epitope retrieval (HIER) at pH 9 (using Bond ER Solution 2) for 30 min before incubation with pre-diluted BRAF V600E mutation-specific primary antibody for 40 min. The Bond Polymer Refine Detection Kit (DS9800 Leica Biosystems, Deer Park, IL) was used to visualize reactivity. Immunoreactions were completed by nuclear counterstaining with hematoxylin.