Immunohistochemistry Protocol for Breast Cancer Biomarkers

As described in previous studies [12 (link)], 3 µm thick tissue sections were cut from the TMA block. After deparaffinization and rehydration using xylene and alcohol graded solutions, respectively, immunohistochemistry (IHC) was performed using Ventana Discovery XT Automated Slide Stainer (Ventana Medical System, Tucson, AZ, USA). Cell conditioning 1 buffer (citrate buffer, pH 6.0; Ventana Medical System) was used for antigen retrieval. Appropriate positive and negative controls were included. Staining of all the IHC markers was assessed using light microscopy. A cut-off value of 1% nuclear staining or more was considered positive for ER and PR [13 (link)]. HER2 staining was interpreted based on the 2018 American Society of Clinical Oncology/College of American Pathologists guidelines [14 (link)]. Only strong and circumferential membranous HER2 expression (3+) was considered positive, while 0 and 1+ HER2 staining was considered negative. Cases with equivocal HER2 expression (2+) were further evaluated for HER-2 gene amplification using silver in situ hybridization.