Cat no 10428 1 ap

The biopsy specimens were cut into 3 um thick sections. Antigen retrieval was performed with 1 mM TRIS-EDTA buffer solution under high pressure (125℃, 103KPa) for 18 min. After inhibition of endogenous peroxidase for 10 min and protein block for 30 min at 37°C, the slides were incubated with rabbit polyclonal antibodies to TGF-α (1:100, ab112030, Abcam, UK), or rabbit polyclonal antibodies to Cytokeratin 18 (CK18) (1:500, ab133263, Abcam, UK), or Rabbit polyclonal antibodies to EGFR at a dilution of (1:100, PAB30732, Bioswamp, Wuhan, China) or Rabbit polyclonal antibodies to MELTF (1:150, Cat No.10428-1-AP, Proteintech, China) overnight at 4°C, then incubated with secondary antibody for 1 h at 37 ℃. The primary antibody was replaced by PBS as negative controls. Quanti cation of immunoreactivity was performed by IPP 6.0, and 4 elds in the endometrial area were randomly selected at a magni cation of × 400 from each slice to determine the mean intensity of optical density (MIOD), the average of the 4 values was considered as the MIOD of the slice. We used immuno uorescence staining for the detection of MELTF protein.

The uterine horns and Ishikawa and HESCs were separately prepared using RIPA (G2002, Servicebio, China) with PMSF (G2008, Servicebio, China). Protein concentration was quanti ed using BCA kit (Cat#P0010S, Servicebio, China). Aliquots of protein samples were separated by SDS-PAGE (G2003, Servicebio, China). The separated proteins were subsequently blotted on to PVDF membrane (G2008G6015-0.45, Servicebio, China) and incubated with primary antibodies to MELTF (1:1000, Cat No.10428-1-AP, Proteintech, China), primary antibodies to LC3B (1:1000, Cat No.18725-1-ap, Proteintech, China), primary antibodies to NLRP3 (1:1000, WL02635, Wanlei, China), primary antibodies to ACSL4 (1:1000, GB113871, Servicebio, China), primary antibodies to FPN1 (1:1000, GB11147, Servicebio, China), primary antibodies to GPX4 (1:1000, Cat No.67763-1-lg, Proteintech, China), primary antibodies to β-actin (1:2000, GB12001, Servicebio, China) or GAPDH (1:2000, GB12002, Servicebio, China). The membrane was then washed and treated with peroxidase-conjugated secondary anti-rabbit antibody (1:5000, GB25301, Servicebio, China) or peroxidase-conjugated secondary anti-mouse antibody (1:5000, GB25301, Servicebio, China).