Identification of trophoblasts and smooth muscle cells and assessment of interstitial trophoblast invasion
Trophoblasts and smooth muscle cells in placenta and uterus were identified by immunocytochemistry. Immunocytochemistry were performed as described previously (Bridgman, 1948; Cotechini et al., 2014; He et al., 2014). Paraffin sections (5 μm) of the rat placentas were prepared, de-waxed, and hydrated, and endogenous peroxides were quenched with 0.3% H2O2. After heat-induced antigen retrieval, the sections were incubated with antibodies against cytokeratin (1:500; Dako, Cat# M515;Clone ID, AE1/AE3) or α-actin (1:500; Abcam, Cat# ab179467; RRID: AB_2737344; Clone ID: EPR16769) overnight at 4℃. Negative controls consisted of the sections in which the primary antibody was substituted with an equal concentration of mouse IgG (DAKO). The bound antibodies were detected with the biotin–streptavidin–peroxidase system (UltraSensitive-SP-kit, MaiXin Biotechnology, Fuzhou, ) using diaminobenzidine (Sigma-Aldrich, ) as chromogen. Counterstaining was performed with hematoxylin.
Assessment of interstitial trophoblast invasion was performed on slides scanned at 20× using Nikon ImageScope system and software. Interstitial trophoblast invasion was calculated by imageJ system as the total area occupied by cytokeratin-positive interstitial trophoblast cells within the mesometrial triangle (MT). Only sections exhibiting the “break through” of interstitial trophoblast cells through the giant cell layer were used to quantify interstitial invasion for consistency (Bridgman, 1948; Cotechini et al., 2014).