Fv500 optical microscope

ADSCs with and without lung cancer cell treatment were collected, centrifuged and fixed in 4% paraformaldehyde for 60 min. Adherent cells and tumour tissues were embedded in paraffin and cut into 4-μm sections. After the tissues were dehydrated in a graded alcohol series, antigen retrieval was performed at 4 °C using 100 μL of a solution containing rabbit monoclonal antibody against human α-SMA/FAP (1:100 dilution; ProteinTech, Chicago, IL). The diluted biotinylated secondary antibody was incubated with the sections for 20 min at 37 °C. Fresh 3,3-diaminobenzidine (DAB) solution was used to visualize the target proteins, and haematoxylin was used as a tissue counterstain. Two observers independently evaluated the expression of target proteins with an Olympus FV500 optical microscope (Olympus, Tokyo, Japan). Image-Pro Plus 5.1 was used to analyse the area and intensity of staining in five random regions (× 200 magnification) to evaluate the protein expression level.

ADSCs (passage 3-6) were analysed via flow cytometry with respect to cellular membrane marker expression using CD105-PECy7, CD73-APC, CD90-FITC, CD34-PE, CD14-APC-Cy7 and CD45-PerCP-Cy5 antibodies (all from eBioscience, San Diego, CA) [43 (link)].
The capacity of ADSCs to differentiate into osteoblasts, adipocytes and chondrocytes was assessed as described [44 (link), 45 (link)]. ADSCs were treated with an Adipogenesis, Osteogenesis and Chondrogenesis Differentiation Kit (Gibco, Invitrogen Corporation, Carlsbad, CA). The medium was changed twice per week. After 3 weeks of differentiation, the ADSCs were stained with Oil Red O and Alizarin Red S. After 4 weeks of differentiation, immunohistochemical staining for type X collagen was performed. The results were recorded using an Olympus FV500 optical microscope (Olympus, Tokyo, Japan).

The antrum tissue specimens were immediately fixed by immersion in 4% paraformaldehyde for 24 h, and then processed for paraffin embedding in a vacuum and cut to a thickness of 4μm. Sections were de-paraffinized in xylene and hydrated in a graded solution of ethanol. After endogenous peroxidase activity was quenched with 3% hydrogen peroxide (H₂O₂) for 10 min and microwaved (750 W) for 5 min, then nonspecific binding was blocked by treatment with normal rabbit serum for 30 min at 37°C. The primary antibodies c-Kit (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) was added to the sections in a moist chamber overnight at 4°C. The slides were washed three times in 0.01mol/l PBS (pH 7.2) and incubated with secondary antibody(HRP-linked polymer antimouse/rabbit IgG)(1:200,ZSGB-Bio,China)for 30 min at 37°C. After washing in PBS three times, the localization of target protein was visualized by incubating the sections for 10 min in freshly prepared 3,3-diaminobenzidine(DAB)solution. The slides were washed again, counterstained in hematoxylin, and then dehydrated. Specificity of the antibody was confirmed by negative control (absence of primary antibody). The slides Positive immunostaining was evaluated at a magnification of x200, using an Olympus FV500 optical microscope (Olympus, Tokyo, Japan).

Approximately 0.5- to 1-cm pieces of uterosacral ligament tissues were collected from the posterior attachment to the cervix during surgery. The tissues were immediately placed in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA), washed with phosphate-buffered saline (PBS) containing 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco), and then cut into small pieces with sterile ophthalmic scissors. The tissues were digested with 10 mg/ml collagenase I (Sigma, St. Louis, MO, USA) for 3 h at 37 °C in 5% CO₂, and digestion was terminated with foetal bovine serum (FBS; Gibco). The digestion liquid was filtered through a 200-mesh screen (75-μm pore size) and then centrifuged at 800 rpm for 5 min. The supernatant was discarded, and the cells were suspended in DMEM containing 10% FBS and cultured in a 25-cm² culture flask. The culture medium was changed every 2 days, and primary fibroblasts were grown to spread across the culture medium for passage. Fibroblasts were used at passage 3–5. The cells were observed with an Olympus FV500 optical microscope (Olympus, Tokyo, Japan).

ADSCs (passages 4–6) were analysed by flow cytometry for cellular membrane expression using CD105-PECy7, CD73-APC, CD90-FITC, CD34-PE, CD14-APC-Cy7 and CD45-PerCP-Cy5 antibodies (all from eBioscience, San Diego, CA).
The capacity of ADSCs to differentiate into osteoblasts and adipocytes was assessed as previously described [19 (link), 20 (link)]. ADSCs were treated with an Adipogenesis Differentiation Kit and an Osteogenesis Differentiation Kit (both from Gibco, Invitrogen Corporation, Carlsbad, CA). The medium was completely changed twice per week. After 3 weeks of differentiation, the ADSCs were stained with Oil Red O and Alizarin Red S. Briefly, ADSCs were washed 3 times with PBS and then fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 20 min at room temperature. The cells were then stained with 0.5% Oil Red O solution for 60 min at room temperature followed by 0.5% Alizarin Red S (both from Sigma-Aldrich, St. Louis, MO) for 20 min at room temperature. The results were assessed from images captured on an Olympus FV500 optical microscope (Olympus, Tokyo, Japan).