Df6096

Cells were lysed with RIPA buffer (CST, Danvers, MA, USA), and 45 ug proteins were run on an 8% SDS-polyacrylamide gel and then transferred onto the PVDF membrane. After 1 hour of blocking with 5% skim milk, the membranes were incubated with primary antibodies overnight at 4°C. The corresponding secondary antibodies were incubated at room temperature for 1 hour the next day. Primary antibodies against IGF-1 (DF6096, 1 : 500), vimentin (AF7013, 1 : 2000), E-cadherin (AF0131, 1 : 500), and β-actin (AF7018, 1 : 2000) were purchased from Affinity Biosciences (Jiangsu, China), with β-actin as an internal control. Subsequently, we washed the membranes (three times for 15 min, each wash) with TBST and incubated them with a goat antirabbit (1 : 3000, Abcam, Cambridge, MA, USA) or antimouse secondary antibody (1 : 3000, Abcam) for 2 h at 26 ± 2°C. An Odyssey infrared imaging system (Odyssey CLx, Biosciences, USA) was used to detect immunoreactivity.

The paraffin sections of human endometrium were baked at 60 °C for 2 h, deparaffined with dimethylbenzenend rehydrated with ethanol series. Antigen retrieval was performed by boiling tissue sections in Tris–EDTA buffer (pH 9.0) (Beijing Solarbio Science & Technology Co., Ltd., China) for 15 min. Next the endogenous peroxidase was removed with 3% hydrogen peroxide and incubated with 5% BSA at room temperature for 1 h. And then, the samples were incubated with rabbit anti-IGF1 (1:100, no. DF6096, Affinity Biosciences, USA) or rabbit anti-IGF1 receptor antibody (1:500, no. 263903, Abcam, USA) or rabbit IgG isotypes at 4 °C overnight. After washing with PBS for three times, the sections were incubated with HRP-labeled secondary antibody at room temperature for 30 min, reacted with 3,3-diaminobiphenylamine (DAB), and finally counterstained with hematoxylin.