Changes in the Temporal and Spatial Expression: MATERIALS AND METHODS(4)

10 Jun

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 in freshly prepared cold Bouin fixative for 24 h essentially as described elsewhere and then embedded in paraffin as detailed by us. Sections (8 ^m) were rehydrated through a series of ethanols, washed in PBS, and treated with 0.1 M glycine in PBS at 22°C for 2 h. To remove endogenous peroxidase activity, tissue sections were quenched in 0.3% (v/v) hydrogen peroxide (Sigma) in methanol at 22°C for 30 min. Samples were blocked for 18 h in a blocking buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% gelatin, 0.05% Tween-20, 0.5% [w/v] powdered milk) at 4°C. The slides were washed in PBS and blocked in normal goat serum (diluted 1:5) for 30 min. The sections were reacted with HBp58 antibody (50 ^g/ml) at 4°C for 18 h and washed three times in PBS. To evaluate specificity of the reaction, some sections were incubated with an equal amount (50 ^g/ml) of preimmune serum.

Primary antibody was reacted with biotinylated affinity-purified anti-rabbit secondary antibody (Vector Laboratory) for 30 min at 22°C. Slides were exposed to the Vectastain ABC reagent, washed in PBS, and reacted for 2 min with equal volumes of 1 ^g/ml diaminobenzidine (Aldrich, Milwaukee, WI) dissolved in 0.1 M Tris, pH 7.2 and 0.1% (v/ v) hydrogen peroxide diluted in PBS. Slides were counterstained with 1% (v/v) methyl green dye in deionized water. At least three separate implantation sites (Days 6-9) and placentas with conceptuses (Days 10, 12) or placentas without conceptuses (Day 14) were examined. Representative sections were photographed using brightfield optics on an Olympus microscope with Kodak Gold (ASA 200) film.