IMMUNOHISTOCHEMICAL BIOMARKERS IN EC 127 5 SUPPLEMENTARY Supplementary S1 Method Detailed information of immunohistochemical analysis Immunohistochemical staining For PR and ER, 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: PR antibody (clone, Pgr 1294 GA090, DAKO, Agilent Technologies, Santa Clara, CA, United States) an ER antibody (clone EP1 GA084, 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 was performed. Thereafter, 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 accomplished by using PowerVision DAB substrate solution (Leica Biosystems, Buffalo Grove, IL, US). For p53 staining, antigen retrieval (30 minutes, pH 6·7) and blocking of endogenous peroxidase with hydrogen peroxide was performed. Subsequently, slides were incubated with p53 antibody (clone DO-7 + BP53-12, dilution 1:600). Powervision+ Poly-HRP was used and visualization was accomplished by using PowerVision DAB substrate solution (Leica Biosystems, Buffalo Grove, IL, US). Counterstaining was performed with hematoxylin, slides were dehydrated and mounted. For the subgroup of patients from the Haukeland university hospital, Bergen, Norway (166 patients), staining protocol differed slightly from the above mentioned staining protocols. Tissue microarrays (TMAs) were constructed for all endometrial biopsies with three tissue cylinders from each case. Microwave antigen retrieval (750 W for 10 and 350 W for 15 min) in Tris–EDTA buffer pH 9 was performed before peroxidase blocking (Dako S-2032) for 5 minutes and incubation with: Oestrogen Receptor α (ER) (Dako M7047) diluted 1:50, Progesterone Receptor (PR) (Dako M3569) diluted 1:150 both for 30 min, purified anti-CD171 antibody clone 14.10 (Biolegend, San Diego, CA, US) diluted 1:100; and tumor protein 53 (p53) (Dako M7001) diluted 1:1000 for 60 min. Subsequently, the
RkJQdWJsaXNoZXIy MTk4NDMw