Anti timp2

Immunocytochemistry of EZH2 and TIMP2 in ovarian cancer cells was conducted as previously described^{11 (link)}. The cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Then, 0.1% Triton-X 100 and 0.3% H₂O₂ were used to break the cell membrane and inhibit endogenous peroxidase activity, respectively. After blocking with 5% BSA at room temperature for 30 min, cells were incubated with primary anti-EZH2 (1:200 dilution, CST, USA) or anti-TIMP2 (1:200 dilution, R&D, USA) antibodies at 4 °C overnight, followed by incubation with biotinylated goat anti-rabbit (for EZH2) or rabbit anti-goat secondary antibody (for TIMP2) (1:100 dilution) for 1 h at room temperature. Normal rabbit IgG (Santa Cruz, USA) was used as a negative control. Immunostaining was performed with diaminobenzidine (DAB). Positive expression was defined as brown-yellow granules distributed in the cytoplasm or nucleus.

Cultured Nestin⁺ and Nestin⁻ BMSCs were seeded on the lower chamber of the 24-well plates, while Sca-1⁺ CECs were plated at 5 × 10⁴ cells/well on the upper compartment of 24-well transwell inserts (8-mm pore size insert, Millipore, Billerica, MA, USA), and the plates were then incubated for 12 h at 37 °C under 5% CO₂. In the control group, no cell was seeded on the lower chamber. After the incubation, the transwell inserts were discarded, and the upper side of the filter was gently swabbed to remove the non-migratory cells. Migrated cells on the lower side of the insert filter were then quickly fixed using 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet for 20 min. Neutralization assays were performed in a transwell system supplemented with anti-TIMP-1 (1.5 μg/ml; R&D Systems), anti-TIMP-2 (3 μg/ml; R&D Systems), and anti-CXCL12 (100 μg/ml; R&D Systems). Microscopic examination was performed and five low-power fields (magnification, × 400) were randomly selected from each chamber. All the experiments in each group were performed in triplicate. The migrated cells were counted by two individuals in a blinded fashion.