In some experiments, sections were processed for immunolocalization of ERa and ERp using a commercially available antibody against ERa (1:80 dilution; Santa Cruz Biotechnology,) or ERp (1:360 dilution; Zymed Laboratories, San Franscisco, CA). The secondary antibody and visualization reagents were used according to the manufacturer’s instructions from the HistoMouse-SP Kit (Zymed Laboratories). As a control for background staining, two methods were used: incubation with secondary antibody, but no primary antibody, or incubation with normal rabbit serum followed by secondary antibody for confirmation of results. Both types of negative controls produced the same result. Six to eight tissue sections were analyzed per slide.
Quantification of immunohistochemical results was carried out with a Zeiss microscope fitted with an MTI CCD72 camera (Dage-MTI, Michigan City, IN) connected to a Macintosh computer, and the NIH Image software program for image analysis. Optical densities were read by the NIH Image analysis software as pixels, which ranged in value from 0 (white) to 255 (black). A standard curve was generated in this grayscale range using a fourth-degree polynomial (R = 1.000) for a relative pixel density range of 0-0.60 units.