In Situ Hybridization of Placental Tissues

Placentae were dissected in cold phosphate buffered saline (PBS) and fixed overnight in 4% paraformaldehyde in PBS at 4°C. After rinsing in PBS, tissues were processed through sucrose gradients (10% in PBS and 25% in PBS) before being embedded in OCT (Tissue Tek). Ten μm sections were cut and mounted on Super Frost Plus slides (VWR) and stored at -80°C. For in situ hybridization, sections were re-hydrated in PBS, post-fixed in 4% paraformaldehyde for 10 minutes, treated with proteinase K (30 μg/ml) for 10 minutes at room temperature, acetylated for 10 minutes (acetic anhydride, 0.25%; Sigma) and hybridized with digoxigenin-labeled probes overnight at 65°C. Digoxigenin labeling was done according to the manufacturers instructions (Roche). Hybridization buffer contained 1× salts (200 mM sodium choride, 13 mM tris, 5 mM sodium phosphate monobasic, 5 mM sodium phosphate dibasic, 5 mM EDTA), 50% formamide, 10% dextran sulfate, 1 mg/ml yeast tRNA (Roche), 1× Denhardt's (1% w/v bovine serum albumin, 1% w/v Ficoll, 1% w/v polyvinylpyrrolidone), and DIG-labeled probe (final dilution of 1:2000 from reaction with 1 μg template DNA). Two 65°C post-hybridization washes (1× SSC, 50% formamide, 0.1% tween-20) followed by two room temperature washes in 1× MABT (150 mM sodium chloride, 100 mM maleic acid, 0.1% tween-20, pH7.5) were followed by 30 minutes RNAse treatment (400 mM sodium chloride, 10 mM tris pH7.5, 5 mM EDTA, 20 μg/ml RNAse A). Sections were blocked in 1× MABT, 2% blocking reagent (Roche), 20% heat inactivated goat serum for 1 hour and incubated overnight in block with anti-DIG antibody (Roche) at a 1:2500 dilution at 4°C. After four 20 minute washes in 1× MABT, slides were rinsed in 1× NTMT (100 mM NaCl, 50 mM MgCl, 100 mM tris pH 9.5, 0.1% tween-20) and incubated in NBT/BCIP in NTMT according to the manufacturers instructions (Promega). Slides were counterstained with nuclear fast red, dehydrated and cleared in xylene and mounted in cytoseal mounting medium (VWR).
For double labeled in situ hybridizations, a fluorescein-labeled probe was generated following the manufacturers instructions (Roche) and added to the hybridization mix along with the DIG-labeled probe. Following NBT/BCIP development of the first probe, the antibody-enzyme conjugate was inactivated by 30 min incubation in 1× MABT at 65°C, followed by 30 min in 0.1 M glycine pH 2.2 at room temperature. The slides were then blocked again in 1× MABT, 2% blocking reagent (Roche), 20% heat inactivated goat serum for 1 hour and incubated overnight in block with anti-Fluorescein antibody (Roche) at a 1:2500 dilution at 4°C. The second signal was developed as previously described but one modification, the substrate INT/BCIP (Roche), producing a brown colour, was used in place of NBT/BCIP. As the INT/BCIP is soluble in alcohols and xylene, slides were counterstained with nuclear fast red and mounted directly under Crystal Mount Aqueous Mounting Medium (Sigma).