Immunohistochemical Profiling of FASN

IHC for FASN was conducted on 4-µm-thick sections from the JHU and PCBN TMAs utilizing a rabbit monoclonal antibody for FASN (Cell Signaling Technology, catalog no. 3180, RRID: AB_2100796) and the Ventana Benchmark immunostaining system (Ventana/Roche; RRID:SCR_021254). To enable antigen retrieval, slides were incubated with CC1 retrieval solution at 100°C for 32 minutes, and the primary antibody was incubated for 40 minutes at a dilution of 1:100. Detection and counterstain reagents used were the OptiView DAB kit (Roche, 760-700; RRID: AB_2833075), hematoxylin and bluing reagents, respectively. The IHC assay was validated using two positive NCI-60 control cell lines (SK-MEL-5, RRID: CVCL_0527 and T47D, RRID: CVCL_0I95) with high FASN RNA expression based on RNA-seq and another NCI-60 cell line with low FASN RNA expression (RXF-393, RRID: CVCL_1673; ref. 31 (link); Supplementary Fig. S1).
FASN-stained TMA slides were scanned at 20x magnification on the NanoZoomer HT Scanner (RRID:SCR_021658). After scanning, each TMA slide was de-cover-slipped, double stained with p63 [mouse monoclonal (4A4), Abcam, #ab735, RRID: AB_305870,1:100, OptiView DAB kit] to distinguish basal cells identifying benign glands and rescanned.