Mab28951

Towbin transfer buffer was used to transfer 10 µg of EV protein separated by SDS-PAGE to a PVDF membrane (Merck Millipore, Germany) at 110 V for 70 min. followed by blockade with 5% skimmed milk powder (BD, USA) for 2.5 h. The membranes were incubated with rabbit polyclonal antibodies anti-CD63 (BBI Life Sciences, D160973; 1:1500, China), anti-CD9 (BBI Life Sciences, D164336; 1:1500, China), anti-TSG101 (ZEN BIO, 381,538; 1:1500, China), anti-calnexin (Abcam, ab75801; 1:1500, UK), and monoclonal antibodies anti-MUC4 (Abcam, ab150381; 1:1500, UK), anti-ACP5 (Abcam, ab191406; 1:1500, UK), mouse monoclonal antibodies anti-MEP1B (R&D Systems, MAB28951; 1:1500, USA) overnight at 4 ℃ followed by incubation with secondary antibody for 1.5 h at room temperature. An Ecl Kit (CWBIO, China) was used to visualize immunoreactive bands and was exposed to EC3 Imaging System (UVP).

Immunohistochemistry was employed to assess the abundance and distribution of MEP1B in various uterine tissue cells during pregnancy, as described in a recent study [23 ]. Briefly, 5 µm thick sections were deparaffinized, blocked for 30 min with 5% bovine serum albumin (BSA), then incubated overnight at 4 °C with antibody (R&D Systems, MAB28951, USA). The primary antibody was replaced with purified corresponding immunoglobulin G as a negative control (NC). Secondary antibodies were used to stain the sections and counterstained with hematoxylin. The images were captured using a Nikon 80i microscope (Nikon, Tokyo, Japan), and the average integrated optical density (IOD) was determined using ImagePro Plus 6.0 software (Media Cybernetics, Silver Spring, GA, USA).