Ab181469

The bones resected from mice were decalcified in a decalcification solution (Merck). Then, the softened bones were immersed in Tissue-Tek O.C.T. Compound (4583, Sakura) and snap-frozen to produce cryosections. Primary antibodies used were as follows: mouse anti-human CD90 antibody (Clone 7E1B11, ab181469, Abcam) and rabbit anti-human Syndecan-1 antibody (Clone SP152, ab130405, Abcam). Alexa Fluor 488-conjugated donkey anti-mouse IgG H&L (ab150105, Abcam) and Alexa Fluor 647-conjugated donkey anti-rabbit IgG H&L (ab150075, Abcam) antibodies were used as secondary antibodies. DAPI (4′,6-diamidino-2-phenylindole; Thermo Fisher Scientific) was used for the staining of the nucleus. Fluorescent signals were detected using the laser scanning confocal microscope (ZEISS LSM 800).

We followed the methods of Sun et al. [17 (link)]. Four percent paraformaldehyde‐fixed, paraffin‐embedded blocks of lung tissues were cut into 4 μm thick sections. The sections were placed on polylysine-coated slides and incubated in a 60 °C oven. The slides were dewaxed using xylene and rehydrated using a gradient of alcohol concentrations. The slides were then placed in a microwave oven until the antigen retrieval solution reached 100 °C for 10 min one and for 2 min four times, cooled to room temperature for 20 min and washed with PBS for 5 min. Primary antibodies against CD90 (Cell Signaling, #13,801, 1:200; Abcam, Ab181469, 1:200), SENP1 (Abcam, Ab236094, 1:200), and alpha-smooth muscle actin (α-SMA) (Abcam, Ab240654, 1:200) were incubated with the slides overnight at 4 °C. Secondary antibodies were incubated with the samples for 1 h at room temperature. Then, the sections were mounted using Fluorescent Mounting Media containing 4′,6-diamidino-2-phenylindole (DAPI) (Abcam). Each tissue section was observed under a confocal laser scanning microscope (Leica SP8, Wetzlar, Germany) at magnifications of 200× and 400×, if necessary.

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.

Rat head samples were collected at required time points, and they were fixed in 10% formalin for at least 24 h. Then they were decalcified in a 10% ethylene diamine tetraacetic acid (EDTA) solution for about 1 month, washed with distilled water, dehydrated, and embedded in paraffin. The tissues were sectioned into slices with 5 μm of thickness and stained by H&E. The immunohistochemical and immunofluorescence studies were conducted by staining the tissue sections using primary antibodies with a dilution of 1:100 and secondary antibodies. The slices were scanned using a tissue section imaging system (Vectra Polaris). The ROIs (region of interest, 1000 × 500 μm, n = 3) of each group were randomly designated, and the number of positive cells was recorded. For immunohistochemistry, the primary antibodies include RUNX2 (ab236639, Abcam), TGFβR2 (66636-1-lg, Proteintech), and Ki67 (ab16667, Abcam, Supplementary Table 2). For immunofluorescence, the primary antibodies include CD90 (ab181469, Abcam) and CD146 (ab75769, Abcam, Supplementary Table 2).