Air si confocal

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.

Immunofluorescence staining was performed on PMSCs in different groups. The cells were grown on poly-l-lysine-coated coverslips in 24-well culture plates. At 80% confluency, cells were fixed in 4% paraformaldehyde for 30 min, rinsed with PBS twice, blocked with 1% bovine serum albumin (BSA) for 30 min, and incubated with rabbit vWF antibody (11,778–1-AP, Proteintech), VEGFR-2 antibody (26,415–1-AP, Proteintech) in a humidified box at 4 °C overnight, followed by washing in PBS three times. FITC-conjugated goat anti-rabbit secondary antibody (Proteintech) or CY3-conjugated goat anti-rabbit secondary antibody (Proteintech) was then added and incubated for a further 30 min. Finally, samples were counterstained with DAPI (Beyotime) in antifade reagent and observed under confocal microscopy (Nikon AIR SI Confocal; Nikon). The control samples consisted of cells without primary antibodies and were used to assess the background fluorescence.