Nuclear ER protein was localized in neonatal uterine tissue sections using the mAb H222 as described previously. Procedures for localization of ER in paraffin-embedded tissue sections were modified from those described by Cartun and Pederson and Papadimitriou et al.. Sections were deparaffinized in xylene, rehydrated through a series of ethanol washes, and rinsed in water. Endogenous peroxidase activity was blocked by incubating sections in 3% H202 for 5 min at room temperature. buy levaquin online
Next, slides were rinsed in water and placed in PBS for 5 min at room temperature. Tissue sections were immersed in 37°C Pronase E solution (0.5 mg/ml; protease type XIV; Sigma) for 8 min. The H222 mAb was applied to tissue sections at a concentration of 5 |xg/ml in PBS, with 1% (w:v) BSA. Visualization of nuclear ER protein was accomplished using the Vecta-Stain Elite ABC kit (Vector Laboratories, Burlingame, CA), which employs the peroxidase enzyme detection system. Negative controls included substitution of irrelevant rat IgG for H222 in uterine sections, elimination of the pronase digestion step, and evaluation of porcine small intestine sections processed identically to uterine tissues stained with the H222 mAb. Nuclear staining intensity (absent, weak, moderate, or strong) for luminal epithelium (LE), glandular epithelium (GE), and stroma (S) was scored independently by two observers. Multiple nonsequential sections from each uterus were examined.