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).