Ab232877

BMSCs were washed with precooled PBS, followed by fixation with 4% paraformaldehyde for 10 min as well as permeability with 0.25% Triton-X100 for 15 min at room temperature. Afterwards, blockage with 1% bovine serum albumin was implemented for 60 min, along with incubation with primary antibody of LIF (1 : 100; ab113262, Abcam), LIFR (1 : 100; ab232877), alkaline phosphatase (ALP; 1 : 200; ab224335), bone sialoprotein (BSP; 1 : 200; ab270605), and osteocalcin (OCN; 1 : 200; ab198228) at 4°C overnight as well as Alexa Fluor® 488- (1 : 200; ab150077; Abcam) or Alexa Fluor® 594- (1 : 200; ab150080; Abcam) conjugated secondary antibody for 1 h. Thereafter, BMSCs were stained with DAPI for 15 min. Images were captured utilizing a fluorescence microscope (Olympus, Japan).

Mice were euthanized via injecting excessive pentobarbital sodium. The cranial bone defect specimens were fixed, decalcified, and dehydrated. Thereafter, the specimens were embedded in paraffin and sectioned into 5 μm, followed by methylene blue and basic fuchsin staining. Sections were immunohistochemically or immunofluorescently stained, and antibodies included CD34 (1 : 100; ab81289; Abcam), LIF (1 : 100; ab113262), LIFR (1 : 100; ab232877), gp130 (1 : 100; ab259927), PI3K (1 : 100; ab32089), p-PI3K (1 : 100; ab182651), AKT (1 : 100; ab8805), p-AKT (1 : 100; ab38449), Nrf2 (1 : 200; ab62352), and GPx-3 (1 : 100; ab256470).

Protein separation was implemented through 12% SDS-PAGE, along with transference to PVDF membrane (Millipore, USA) as well as incubation with defatted milk. Afterwards, the PVDF membrane was subjected to immunoblotting following the indicated primary antibodies: Kelch-like ECH-associated protein 1 (Keap1; 1 : 500; ab119403; Abcam), nuclear factor-erythroid 2-related factor 2 (Nrf2; 1 : 200; ab62352), LIF (1 : 1000; ab113262), LIFR (1 : 1000; ab232877), gp130 (1 : 1000; ab259927), PI3K (1 : 1000; ab32089), p-PI3K (1 : 500; ab182651), AKT (1 : 500; ab8805), p-AKT (1 : 500; ab38449), hypoxia-inducible factor 1α (HIF-1α; 1 : 100; ab51608), superoxide dismutase 1 (SOD1; 1 : 5000; ab51254), catalase (1 : 1000; ab52477), glutathione peroxidase 3 (GPx-3; 1 : 1000; ab256470), and GAPDH (1 : 2000; ab59164). Incubation with a secondary antibody was implemented for 1 h. Protein was developed with an enhanced chemiluminescence approach.