Ovary-Independent Estrogen Receptor Expression: MATERIALS AND METHODS(6)

17 Jan
2013

MATERIALS AND METHODS(6)

Slides of porcine endometrium developed to display in situ-hybridized ER antisense and sense cRNAs were examined at XlO, X20, and X40 by darkfield microscopy on the same microscope equipped with an Olympus darkfield condenser. Darkfield images were captured from a silicon intensified target (SIT) camera (model 68; Dage/MTI). Both brightfield and darkfield images were background subtracted and were processed digitally using constant parameters with a commercial image-processing package (Image-Pro Plus; Media Cybernetics, Bethesda, MD) on a Pentium-based microcomputer. Images were printed on a high-resolution laser printer (Tektronix, Wilsonville, OR). Great care was taken to keep image capture and processing conditions identical for controls and corresponding experimental conditions, and between iterations of the image capture and processing effort for related experiments. All images were corrected for the same background level by manual adjustment of the camera black level to the same image bit-value by comparison against a pseudocolor standard, and the gain and the SIT camera-intensified target voltage were maintained at the same value for all related images to ensure an accurate representation of the relative local image intensities. Thus, all images of a given type (i.e., all darkfield images and, separately, all brightfield images) were directly comparable to each other, and all images shown herein represent the relative local image signal realistically.

Statistical Analyses

Data for uterine weight and endometrial thickness were subjected to least-squares analysis of variance using General Linear Models procedures. The statistical model included effects of age, treatment (ovariectomy), and their interaction. Tests of significance were based on expectations of the error mean squares. Least-squares regression analyses and tests for heterogeneity of regression were performed to determine whether patterns of change in uterine wet weight or endometrial thickness differed between intact and OVX gilts in association with advancing postnatal age. Data are presented as least-squares means with standard errors. ventolin inhaler

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