Breast Cancer Tissue Microarray Analysis

Breast cancer tissue microarray slides (Cat No. BR 243v, BR 246a) were purchased from US Biomax (Rockville, MD, USA). The slides were stained by anti-ERRβ antibody at 1:50 dilution (sc-68879, Santa Cruz, Dallas, TX, USA) and were further processed using ABC system (Vector Laboratories, Bulingame, CA, USA) as described previously [38 (link)]. The images were captured under Leica microscope (Wetzlar, Germany) using LAS EZ software version 2.1.0. The slides were examined and scoring was done by an experienced pathologist. The intensity score was calculated based on staining for ERRβ and was assigned from 0 to 3 (0 indicating no staining; 1+ weakly stained; 2+ moderately stained and 3+ strongly stained positively). The percentage of positively stained cells were scored as follows, 0- no positive staining; 1+, 1–25% positively stained cells; 2+, 26–50% positively stained cells; 3+, 51–70% positively stained cells; 4+, > 70% positively stained cells. The composite score was calculated using both intensity score and the percentage of positive cells as it is a product of both scores. The composite score range was given from 0 to 12. The samples scored < 3 were considered as low categorized; 3–5 moderately categorized; ≥ 6 highly categorized. The graph was plotted using composite scores using GraphPad Prism version 6.01.