Quantitative immunofluorescent estimation of estrogen receptor β expression in human solid tumors using flow cytometry

The optimization of an immunofluorescent analysis for estrogen receptor β (ER-β) expression estimation was performed using flow cytometry (FC) and monoclonal antibodies (Abacam Co.) specific to the N-terminal domain of the human ER-β protein (clone 14C8). We have shown the possibility of cell fixation in a 4%-formaldehyde solution while varying the number of cells in the suspension, which allows one to examine tumor specimens obtained both during surgery and endoscopic examination of patients and tumor biopsies. Overnight incubation for (15-20 h), with primary antibodies and incubation with secondary antibodies for 1.5 h is recommended for use in clinical practice because of it optimizes the sensitivity of the method, reduces antibody expenses, and is convenient. Breast cancer cells of the MCF-7 line as a reference culture and a range of antibody concentration of 0.06, 1.2, and 2.5 μg/ml are recommended as controls for the antibody activity. The range of antibody concentration of 1.2, 2.5, and 5 μg/ml is recommended for use in the quantitative estimation of ER-β expression in human solid tumors, which covers both the linear dependence region of fluorescent specific staining on antibody concentrations and the plateau region of staining all the cells in the investigated cell suspension and, as a consequence, in the examined tumor. © 2011 Allerton Press, Inc.