Cell pellets were fixed in 4% paraformaldehyde overnight at room temperature and processed for paraffin embedding using Leica ASP6025 tissue processor (Leica Biosystems). Freshly cut 5-μm paraffin sections were stained for Renin polyclonal (5 μg/ml; #AF4090, R&D Systems) for 1 hour and using 10 min of 1:200 Tyramide Alexa Fluor 488 detection (#T20948, Life Technologies) on Leica Bond Protocol F. The sections were pretreated with Leica Bond ER2 Buffer (Leica Biosystems) for 20 min at 100°C before staining.

First-trimester placental villi tissues were fixed with 10% neutral buffered formalin (Azer Scientific) at room temperature for 24 hours. Samples were transferred to 70% ethanol, followed by paraffin embedding. Freshly cut 5-μm paraffin sections were stained for sequential double immunofluorescence on Leica Bond RX (Leica Biosystems) with Vimentin mouse monoclonal (0.05 μg/ml; #VP-V684, Vector Labs) for 1 hour and using 10 min of 1:200 Tyramide Alexa Fluor 647 detection (#T20951, Life Technologies) on Leica Bond Protocol F, followed by Renin polyclonal (0.2 μg/ml; catalog no. AF4090, R&D Systems) for 1 hour and using 10 min of 1:200 Tyramide Alexa Fluor 488 detection (#T20948, Life Technologies) on Leica Bond Protocol F. The sections were pretreated with Leica Bond ER2 Buffer (Leica Biosystems) for 20 min at 100°C before each staining. Images were recorded on the Olympus VS110 and processed using Visiopharm Integrated Systems software.

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