Bx51 upright fluorescence microscope

The effect of Lefty-1 on NRK-49F cell proliferation was also evaluated using the 5-Bromo-2-deoxyuridine (BrdU) incorporation assay. Briefly, cells were seeded on coverslips at a density of 1.0×10⁶ cells/well in six-well plates. Cells were incubated for 48 hours, as previously described, and then pulsed with BrdU (10 μg/mL) for 24 hours. Cells were fixed with 4% paraformaldehyde for 15 minutes, and the DNA was denatured with 2 N of HCL at 37°C for 30 minutes, following by washing with PBS. Endogenous peroxidase activity was eliminated using 3% H₂O₂ in PBS for 20 minutes. After washing with PBS three times, cells were permeabilized with 0.3% Triton X-100 buffer at room temperature for 30 minutes, and nonspecific binding was blocked with 1% goat serum at 37°C for 1 hour. Incorporated BrdU was detected using a rabbit monoclonal anti-BrdU antibody overnight, and then the cells were stained with CY3-conjugated secondary antibody. Finally, nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). The immunofluorescent images were visualized by an Olympus-BX51 fluorescence upright microscope (Olympus Corporation, Tokyo, Japan). The percentage of BrdU-positive nuclei was determined by the proportion of the number of BrdU-positive cells to total NRK-49F cells.

Indirect immunofluorescence was used on control and Lefty-1-treated NRK-49F cells on coverslips. The cells were washed with PBS at 37°C and fixed in 4% paraformaldehyde for 10 minutes, and then permeabilized using 0.5% Triton X-100 buffer for 5 minutes at room temperature. Afterward, the cells were extensively washed thrice with PBS. These slides were blocked with 1% bovine serum albumin (BSA) in PBS for 20 minutes at 4°C and then incubated with the primary antibodies as described above. The following polyclonal antibodies were used: antifibronectin (1:100), anticollagen I (1:100), anti-α-SMA (1:100), and anti-PCNA (1:100). To visualize the primary antibodies, the cells were stained with fluorescein isothiocyanate or cyanine dye-labeled secondary antibodies (1:100). Stained cells were mounted using the antiquenching fluorescence mounting medium (Wuhan Good Biotechnology CO., LTD, Wuhan, Hubei, People’s Republic of China) and viewed using an Olympus-BX51 fluorescence upright microscope (Olympus Corporation). Quantitation of immunofluorescence staining was carried out on coded cell coverslips as the integrated option density value.