Fluorescence in situ hybridization protocol

Fluorescence in situ hybridization was performed as previously described (Yuan et al., 2019 (link)). In brief, implantation sites from three individual animals in each experimental group were collected. Frozen sections (12 μm) from three implantation sites from different females in each group were mounted onto poly-L-lysine-coated slides and fixed in 4% paraformaldehyde in PBS. Following acetylation and permeabilization, slides were hybridized with the DIG-labeled Prl8a2 probes at 55°C overnight. After hybridization, slides were then washed, quenched in H₂O₂ (3%), and blocked in blocking buffer (1%). Anti-Dig-peroxidase was applied onto hybridized slides and color was developed by Tyramide signal amplification (TSA) Fluorescein according to the manufacturer’s instructions (PerkinElmer). Cytokeratin 8 (Iowa hybridoma bank, 1:100 dilution) was used to outline epithelial cells. Images presented are representative of three independent experiments.