Hrp labeled goat anti mouse secondary antibody

The total protein was extracted from villi and decidual tissue by conventional method. The bicinchoninic acid assay (BCATM Protein Assay Kit, Pierce, USA) was used to quantify the protein. Then, the protein was subjected to 10% SDS-PAGE electrophoresis (Mini-Protean-3, Bio-Rad, USA), and the membrane was blocked with 5% skim milk. Mouse anti-human LRH-1 primary antibody (1: 1000, Abcam, USA) was added and incubated at 4°C overnight. After that, the membrane was washed 3 times with TBST for 10 min for each time. HRP-labeled goat anti-mouse secondary antibody (Abcam, USA) diluted with TBST (1: 500) was added and incubated at room temperature for 1 h. After that, ECL was added for exposure. β-actin was used as the endogenous reference. ImageJ2.1 (National Institutes of Health, USA) software was used to analyze the results. The relative expression levels of LRH-1, CYP19, and P450scc proteins were expressed by the ratios of the values of LRH-1, CYP19, and P450scc protein to that of β-actin.