Posts Tagged ‘Implantation

Characterization of H/358 Antibody by Western Blotting We tested the specificity of the Hp58 antibody by Western blotting. Induction of protein expression was monitored by SDS PAGE (Fig. 3A). The induced GST-Hp58 fusion protein migrated at a molecular mass of 35 kDa consistent with the predicted amino acid sequence of the coding region of the […]

Preferential Amplification of the Rat H/358 cDNA from the Implantation Site Complementary DNAs prepared from total RNA that was isolated from the implantation sites (Evans blue dye positive) and the intersite regions of the uterus at Day 5 of pregnancy were amplified using RNA fingerprinting. One PCR product unique to the implantation site was excised […]

Ribonuclease Protection Assays The rat Hp58 cDNA that was isolated and cloned from the RNA fingerprinting analysis was restricted with HindIII and transcribed in vitro using T3 polymerase to produce single-stranded RNA probes using the Riboprobe kit (Pro-mega Biotech, Madison, WI) and [a-32P]CTP (800 Ci/ mmol; ICN). To adjust for assay variability a rat glyceral-dehyde […]

Localization of H/358 in the Postimplantation Uterus and the Maternal and Fetal Placenta Implantation sites from pregnant rats at Days 6-9 of pregnancy were fixed in 4% (w/v) paraformaldehyde (Fisher Scientific, Hanover Park, IL) in PBS and embedded in paraffin using methods standard in our laboratory. Placental tissues (Days 10, 12, and 14) were fixed […]

Antibody Specificity To assess antibody specificity, the rat Hp58 cDNA was expressed as a fusion protein and reacted with the Hp58 antibody on Western blots. The Hp58 cDNA that had been isolated and cloned from the RNA fingerprinting analysis was amplified by PCR using Hp58 gene-specific primers. The resulting PCR product was subcloned in frame […]

Northern Blot Analysis Samples of total RNA were dried under vacuum centrifugation, suspended in denaturing solution, and loaded onto 2.2 M formaldehyde-1% agarose gels. The RNA size markers (RNA ladder; Life Technologies, Gaithersburg, MD) were electrophoresed on the same gel to determine transcript size. The RNA was transferred by diffusion onto nylon membranes (Micron Separation, […]

Animals Mated female Sprague-Dawley rats were housed on a 14L:10D cycle at the University of Missouri-Kansas City and provided rat chow and water ad libitum. The presence of sperm in the vagina was determined by the vendor (Ta-conic, Germantown, NY) and was counted as Day 1 of pregnancy. Animals were treated in accordance with the […]