Ovary-Independent Estrogen Receptor Expression: MATERIALS AND METHODS(5)

16 Jan
2013

In Situ Hybridization

In situ hybridization (ISH) was performed, using [35S]UTP-labeled (specific activity: 800 Ci/mmol; Amersham, Arlington Heights, IL) antisense and sense cRNA probes produced by in vitro transcription from the EcoRI-and BamHI-linearized 0ER8 cDNA template, with a MaxiScript kit (Ambion, Austin, TX). T7 and SP6 RNA polymerases were used to transcribe antisense and sense probes, respectively. Procedures were essentially identical to those previously described. Each slide included tissue sections that received either radiolabeled sense (negative control) or antisense cRNA probe. Hybridization was carried out overnight at 55°C. After posthybridization processing, slides were dipped in NTB-2 liquid photographic emulsion (Eastman Kodak) and exposed at 4°C for 12 wk. Slides were developed at 15°C using D19 developer (Eastman Kodak) and counterstained with mercury-free Harris modified hematoxylin with acetic acid (Fisher Scientific, Pittsburgh, PA). birth control pills

Light Microscopy and Digital Imaging

Brightfield images of porcine endometrial tissues stained with hematoxylin and eosin, and for nuclear ER protein, were obtained using XlO, X20, or X40 planachromat objectives on a conventional compound light microscope (model BHS; Olympus, New York, NY). Brightfield images were captured directly to a digital image capture board (model Flashpoint; Integral Technologies, Indianapolis, IN) using an instrumentation-grade Newvicon camera designed for high-quality video-enhanced microscopy (VE1000; Dage/MTI, Michigan City, IN).

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