Immunohistochemistry was performed as previously described [36 (link), 37 (link), 39 (link)]. Placentas from pregnant mice at E18.5 were harvested, formalin fixed, dehydrated, and embedded in paraffin. Tissue sections, 5 μm in thickness, were cut and deposited on poly-lysine-coated slides. To improve antigen recognition, the sections were microwaved in 10 mM citrate buffer (pH 6.0) for 10~12 min. They were blocked with 10% donkey serum and then incubated with primary antibodies at 4 °C overnight. Primary antibodies were applied in this study: CD117 (R&D Systems, Cat. AF1356, 1:50), cytokeratin 7 (CK7; Abcam, Cat. ab181598, 1:1000), Ki67 (Dako, Cat. M7249, 1:100), CD34 (Abcam, Cat. ab8158, 1:50), CD45 (Abcam, Cat. ab10558, 1:50), CD31 (Abcam, Cat. ab28364, 1:50), vimentin (Abcam, Cat. ab92547, 1:200), E-cadherin (Abcam, Cat. ab53033, 1:50), cardiac troponin I (cTnI, Abcam, Cat. ab47003, 1:200), SPC, and Rho (see “Immunocytochemistry ”). Finally, species-matched secondary antibodies conjugated with fluorescence were applied at 37 °C for 1 h. Nuclei were stained with DAPI. A 3,3′-diaminobenzidine system was also carried out for CD117 staining. Images were analyzed using a fluorescence or light microscopy or a confocal microscopy system (Olympus).
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