Ab51054

The cultured endometrial cells were inoculated on sterile coverslips at 1 × 10⁵/ml and cultured in an atmosphere of 5% CO₂ at 37°C for 5 days, washed three times with PBS, soaked in methanol at −20°C for 10 min, and blocked with 5% normal goat serum (NGS) at 37°C for 30 min. The cells were incubated overnight at 4°C with a primary antibody: keratin (ab51054; 1 : 100; Abcam, Cambridge, United Kingdom) for glandular epithelial cells, vimentin (ab254015; 1 : 100; Abcam) for stromal cells, anti-LHR (sc-25828; 1 : 50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), annexin IV (3175–1; 1 : 500; RabMabs, Burlingame, CA, USA), and VEGF (sc-705; 1 : 50; Santa Cruz). PBS, instead of the primary antibody, was used for negative control. The secondary antibody was HRP-linked anti-mouse IgG (7076S; 1 : 1500; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at room temperature. The staining was revealed with DAB for 3–5 min. The cells were counterstained with hematoxylin for 30 s. The cells were dehydrated and mounted with a mounting medium (Wuhan Boshide Biotechnology Co., Wuhan, China). Examination and photography were performed using an inverted microscope (Carl Zeiss GmbH, Oberkochen, Germany).

Cells were cultured in four well TEK slides (Fisher) and fixed for 10 min using freshly made 4% neutral-buffered formalin. Primary rabbit monoclonal antibodies to keratin 14 (K14, ab51054, Abcam) and mouse monoclonal antibody to K18 (ab7797, Abcam) were used for double staining. Secondary donkey anti mouse antibody (ab150105, Abcam) with Alexa 488 tag and secondary goat anti rabbit antibody (ab150080, Abcam) with an Alexa 594 tag were used for visualization. Negative controls consisted of irrelevant primary antibodies including rabbit monoclonal isotype control (ab172730, Abcam) and mouse monoclonal isotype control (ab170190, Abcam). Primary mouse monoclonal antibody to K17 (ab188859, Abcam), mouse monoclonal antibody to MMP7 (sc-515703, Santa Cruz) and rabbit monoclonal antibodies to p63 (#13109S, Cell Signaling) were used for single staining. The dilution ratio for all primary antibodies was 1:300 and the dilution ratio for all secondary antibodies was 1:500. The gain and exposure time of the fluorescence microscope were adjusted to show positively stained cells while negative control cells had no staining. Staining percentage was calculated by dividing the positively stained cell number by the total cell number. Staining intensity was measured by ImageJ and the negative control was used to subtract background.