Tcs nt 4d confocal microscope

Cells were cultured for 48 hours on glass coverslips in 6-well plate and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 5 min at room temperature, incubation with primary antibody for one hour, wash with 1% Triton X-100, and incubation with Alexa Fluor-conjugated secondary antibodies for one hour in the dark. The coverslips were stained with Hoechst 33342 (H3570, Invitrogen, 1:3000) before being mounted with VECTASHIELD mounting medium (H-1000, Vector laboratories). Images were captured by Leica TCS-NT 4D confocal microscope. Z stacks were collected with a spacing of 1 μm. Antibody and dilutions used in the studies: anti-Flag (14793S, Cell signaling, 1:100), anti-Lamin B1 (68591S, Cell signaling, 1:100), anti-STAT3 (MA1–13042, Thermo Fisher Scientific, 1:100). Alexa Fluor 488 Goat anti-Rabbit (A11008, Life technologies, 1:5000), Alexa Fluor 594 Goat anti-Mouse (A11032, Life technologies, 1:5000). The fluorescence-intensity profile along the Z-axis from confocal Z-stacks were shown. The fluorescence intensity of nuclear localized STAT3 was quantified using the confocal software to define the selected ROI (Region of Interest) area based on nuclear DAPI signal. More than 200 cells were quantified in at least three independent experiments.

The cell proliferation assay was performed using Invitrogen Click-iT EdU Imaging kit (C10337, Invitrogen). At 24 hours post transfection, OVCAR8 cells were split and seeded on glass coverslips in 6-well plates. After 24 hours incubation, the cells was treated with10 μM EdU for 16 hours. Then cells were fixed with 4% paraformaldehyde in PBS for 10 min, followed by 0.5% Triton X-100 permeabilization for 20 min at room temperature. After Click reaction for EdU detection, immunofluorescent staining was carried out using anti-HA antibody. Hoechst 33342 was diluted in PBS (1:2000) for DNA staining. Images were captured by Leica TCS-NT 4D confocal microscope and analyzed to determine the percentage of the EdU positive cells. The statistical significance of the data was determined by GraphPad Prism 5.0 software.