Ab266423

The collected placental tissues were washed three times in PBS; then, tissues were embedded in paraffin and sliced into 5 μm sections. After deparaffinization and antigen retrieval, sections were rehydrated in PBS for 15 minutes and then treated with PBS containing 0.1% of Triton X-100 and 1% of SDS for 4 minutes. Sections were washed in PBS for 5 minutes and blocked in 1% of BSA for 15 minutes. Sections were then incubated overnight in a humid chamber at 4°C with anti-TLR4 (1 : 100, 19811-1-AP, Proteintech), anti-SHH (1 : 100, 20697-1-AP, Proteintech), anti-SMO (1 : 200, ab266423, Abcam), anti-Gli1 (1 : 100, A14675, Abclonal), and anti-CD90 (1 : 200, ab181469, Abcam). Sections were washed in PBS three times for 5 min. Appropriate secondary antibodies were then applied for 1 h in the dark. Nuclei were stained with DAPI. PMSCs were labeled with anti-CD90 [53 (link)]. The sections were placed on the glass slides and were imaged by confocal microscopy (Nikon AIR SI Confocal; Nikon).
Different groups of PMSCs were seeded on coverslips and were fixed by 4% paraformaldehyde for 30 min. After blocking with 1% bovine serum albumin, PMSCs were incubated with anti-SMO (1 : 200; ab266423; Abcam) overnight. Then, PMSCs were incubated with secondary antibody, following with DAPI. Images were captured with a microscope and were analyzed by ImageJ.
All experiments were performed three times.

Total proteins were extracted from cells with lysis buffer and were incubated for 15 min at 95°C. Then, proteins were subjected by 10% SDS-PAGE and then transferred to PVDF (polyvinylidene fluoride) membranes (0.45 μm pore size; Millipore, MA, USA). The membranes were blocked with quick blocking buffer (P0252; Beyotime, China) for 10 min and then incubated with the anti-TLR4 (1 : 1,000; sc-293072; Santa Cruz), anti-SHH (1 : 1,000; 20697-1-AP; Proteintech), anti-SMO (1 : 2,000; ab266423; Abcam), anti-Gli1 (1 : 1,000; A14675; ABclonal), anti-p16 (1 : 1,000; A11651; ABclonal), anti-p53 (1 : 1,000; A19585; ABclonal), anti-MMP9 (1 : 1,000; 10375-2-AP; Proteintech), or anti-β- actin (1 : 4,000; 20536-1-AP; Proteintech) overnight at 4°C. The membranes were washed with TBST three times and then incubated with a secondary anti-rabbit antibody (1 : 4,000; GB23303; Servicebio) or anti-mouse antibody (1 : 4,000; GB23301; Servicebio) for 1 h. Finally, the membranes were visualized in chemiluminescence method (WVKLS0500; Millipore). All experiments were performed three times.