PURE AND MIXED CLEAR CELL CARCINOMA 97 4 SUPPLEMENTARY Supplementary A. Detailed information on immunohistochemical staining For ER and PR, antigen retrieval (97 ºC for 30 minutes in Tris/EDTA buffer pH 9 [Envision FLEX Target Retrieval Solution High pH, DAKO, Agilent Technologies, Santa Clara, CA, United States]) and blocking of endogenous peroxidase with hydrogen peroxide were performed. Subsequently, slides were incubated with: ER antibody (clone EP1 GA084, DAKO, Agilent Technologies, Santa Clara, CA, United States) and PR antibody (clone, Pgr 1294 GA090, DAKO, Agilent Technologies, Santa Clara, CA, United States). Envision FLEX/HRP (DAKO, Agilent Technologies, Santa Clara, CA, United States) was used and visualization was performed using Envision FLEX DAB+ Chromogen (DAKO, Agilent Technologies, Santa Clara, CA, United States). For L1CAM, EDTA (95 ºC for 10 minutes in Tris-EDTA buffer pH 9) and blocking of endogenous peroxidase with hydrogen peroxide were performed. Subsequently, slides were incubated with: L1CAM antibody (purified anti-CD171, clone 14.10, Biolegend, San Diego, CA, US, dilution 1:100). Powervision+ Poly-HRP was used and visualization was performed using PowerVision DAB substrate solution (Leica Biosystems, Buffalo Grove, IL, US). Immunohistochemical analysis of the mismatch repair (MMR) proteins PMS2 and MSH6 was performed. In short, blank 4μm formalin-fixed, paraffin-embedded (FFPE) sections were cut on Superfrost+ glass slides. After antigen retrieval with EnVision FLEX High pH Target Retrieval Solution, and blocking of endogenous peroxidase with hydrogen peroxide, all slides were incubated with anti-MSH6 (clone EPR3945 1:400, Abcam, Cambridge, UK) or anti- PMS2 (clone A16-4 dilution 1:20, BD Biosciences, San Jose, CA). Subsequently, they were incubated with EnVision FLEX and visualized with High pH visualization system according to the manufacturer’s instructions for use. Counterstaining was performed with hematoxylin, and the slides were dehydrated and mounted.
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