Evaluation of seven oestrogen receptor β antibodies for immunohistochemistry, western blotting, and flow cytometry in human breast tissue

Two oestrogen receptors, ERalpha and ERbeta, exist. While much is known about ERalpha, the role of ERbeta is still undefined, especially at the protein level. The aim of this study was to determine the utility of seven ERbeta antibodies (14C8, 8D5, PA1313, PPG5/10, N19, 9.88, and D7N) raised against different domains of ERbeta in three commonly used laboratory applications, namely, immunohistochemistry, western blot, and flow cytometry, using human breast material. For immunohistochemical analysis of frozen material, PA1313 and D7N gave stronger and more specific signals than 14C8, 8D5, and PPG5/10. In paraffin sections, 14C8, closely followed by PPG5/10, gave by far the most superior nuclear immunoreactivity, compared with the other antibodies tested. In general, flow, cytometry results mirrored the immunohistochemistry data for paraffin sections, with antibodies ranked 14C8 > 8D5 greater than or equal to PAI-313 > PPG5/10 > D7N. For western blotting, 8135 and D7N yielded the strongest and most consistent bands, with weaker bands seen with the others. It is concluded that ERbeta protein can be detected using specific antibodies. However, there is onsiderable variation between the specificity and application of these antibodies, highlighting the fact that careful optimization is required when selecting an antibody for use in a particular laboratory technique. Copyright (C) 2002 John Wiley Sons, Ltd.