Anti fth1

A total of 60 μg of protein extract was resolved on 15% SDS-PAGE and then transferred to a nitrocellulose membrane by electroblotting [32 (link)]. Non-specific reactivity was blocked in nonfat dry milk in TTBS 1X [5% (w/v) milk in TBS 1X (pH 7.4) and 0.1% Tween [20 (link)] for 2 h at room temperature. The membrane was incubated with specific primary antibodies anti-FTH1 (sc-376594 Santa Cruz, Santa Cruz, CA, USA), Anti-Flag M2 (F-1804 Sigma Aldrich) and anti-PRDX6 (ab59543 Abcam, Cambridge, UK) overnight at 4 °C. After incubation, and the membranes were washed three times with TTBS 1X for 10 min and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. According to the manufacturer’s instructions. The membranes were washed three times with TTBS 1X, and signals were visualized by ECL-Western blot detection reagents (Santa Cruz Biotechnology, Dallas, TX, USA [45 (link)]).

For the immunofluorescence staining of human decidual tissues, the deparaffinized and rehydrated slides were boiled in 10 mM citrate buffer (pH 6.0) to enable antigen retrieval. Then, the slides were blocked with 10% normal donkey serum for 1 h at room temperature and incubated with primary antibodies which included anti‐TNFα (Abclonal, China), anti‐FTH1 (Santa Cruz, USA), anti‐TNFR1 (Abcam, USA), anti‐TNFR2 (Proteintech, China), anti‐NF‐κB (Proteintech, China) and anti‐caspase3 (Abclonal, China) for 4℃ overnight. After that, the slides were washed by 1× PBS three times for 10 min each time, followed by 1‐hour room temperature incubation of corresponding fluorescent secondary antibodies (AlexaFluor 594 anti‐Rabbit, AlexaFluor 488 anti‐Mouse, Invitrogen, USA) and 4,6‐diamidino‐2‐phenylindole (DAPI). The slides were scanned and imaged by a digital slice scanner (Pannoramic MIDI, 3DHIESTECH, Hungary).
For the immunofluorescent staining of cultured cells, the slides were fixed with 4% paraformaldehyde and punched with 0.3% Triton X‐100 in PBS for 15 min. After that, the slides were blocked with 5% BSA and incubated with primary antibodies including anti‐NF‐κB (Proteintech, China). The following steps were consistent with the immunofluorescent staining of decidual tissues. The slides were observed and imaged by a laser scanning confocal microscope (Olympus, Japan).