Hla g

The placental tissues were embedded in paraffin after being treated with 4% paraformaldehyde. The tissues were then cut into slices of 3–5 μm thickness. The tissue slides were serially drenched in ethanol and dewaxed in xylene. For antigen retrieval, the slides were then submerged in sodium citrate buffer and warmed in a microwave for 15 min at 50 °C and then 4 min at 100 °C. Following the use of an autofluorescence quenching agent (Sevicebio, Wuhan, China), goat serum was used to block the sample for an additional hour at room temperature. The appropriate antibodies were used to hatch the slides over night at 4 °C. The specific antibodies used in this study were CCL19 (1:50; ABclonal Technology, Wuhan, China); CCL21 (1:100; Aifang Biological, Changsha, China); CCR7 (1:50; ABclonal Technology, Wuhan, China); CK7 (1:50; Aifang Biological, Changsha, China); and HLA-G (1:50; ProteinTech, Wuhan, China). During the following day, the slides were incubated with fluorescein-conjugated secondary antibodies (1:100; Abbkine Biotechnology, Wuhan, China). 4′,6-Diamidino-2-phenylindole was used to stain the nuclei (DAPI, Servicebio Biotechnology, Wuhan, China).

To prepare whole‐cell protein lysates, cells at 90% confluence were washed in PBS before incubation with RIPA lysis buffer. Equal protein was loaded onto 10% SDS‐PAGE gels, transferred onto nitrocellulose membranes and blocked with TTBS containing 5% fat‐free dry milk. The membranes were incubated at 4°C overnight with the primary antibodies: uPA and uPAR (Abcam), p‐c‐FOS (Ser³²), p‐c‐JUN (Ser⁷³), c‐FOS, c‐JUN, Akt, p‐Akt (Tyr³⁰⁸), PDK and p‐PDK (Ser²⁴¹) (Cell Signaling Technology) and CK‐7, vimentin, N‐cadherin, E‐cadherin, poFUT1, HLA‐G and GAPDH (Proteintech). Next, the membranes were incubated with HRP‐conjugated goat anti‐rabbit IgG, HRP‐conjugated goat anti‐mouse IgG or HRP‐conjugated streptavidin for 1 hour. An enhanced chemiluminescence detection system (Bio‐Rad) was used to visualize immunoreactive bands.