Eclipse ti microscope system

Live-cell imaging was performed on a Nikon Eclipse Ti Microscope System (Nikon Instruments, Melville, NY) at 37°C and 5% CO₂. Images were captured using an electron-multiplying charge-coupled device camera (Photometrics Evolve 512; Photometrics, Tucson, AZ) and a Plan Apo VC 60× H objective (numerical aperture [NA] 1.40). Diffraction-limited fixed-cell imaging was performed with a Zeiss 780 (Carl Zeiss). Superresolution imaging was performed using a Zeiss 880 Airyscan microscope and a 63×/1.40 NA Plan-Apochromat Oil DIC M27 objective lens (Carl Zeiss) and processed at setting 6. Image processing was performed in ImageJ (http://imagej.nih.gov/ij/), including cropping and average/maximum intensity projections. Photobleaching experiments were performed at 37°C and 5% CO₂ on a Nikon Eclipse Ti Microscope System equipped with a Galvo miniscanner to allow photobleaching and subsequent spinning-disk confocal imaging. TIRF images were acquired with an Apo TIRF 100× objective.

The xenograft tumors were fixed in 2% paraformaldehyde for 24 hours. The paraffin-embedded tissue was cut into 5-µm sections. The sections were deparaffinized, rehydrated, and then subjected to high pressure for antigen retrieval (pressure, 15PSI; temperature, 121 °C) in EDTA antigen retrieval buffer (1 mM EDTA; pH 8.0). Endogenous peroxidase was inactivated with 3% hydrogen peroxide, and 1% bovine serum albumin (Sigma) was used to block non-specific binding for 1 hour. Anti-Ki-67 (1:200; Abcam), anti-MCM2 (1:200; Abcam), and anti-PCNA (1:1,000; Cell Signaling Technology) antibodies were used to detect the expression levels of the corresponding proteins in the mouse xenograft tissues. These antibodies were the same as those used for western blotting and have been described previously. Tissue samples were incubated in a primary antibody solution overnight at 4 °C. The secondary antibody Biotin Conjugated AffiniPure Donkey Anti-Rabbit IgG (1:200; Wuhan Boster Biological Technology) was incubated for 1 hour . Finally, the tissue slices were visualized with a Nikon ECLIPSE Ti microscope system (Nikon Corporation), and the images were processed using Nikon software (NIS-Elements v.AR4.10, Nikon Corporation). IHC Profiler (an ImageJ plugin) was used to perform automatic quantitative analysis in line with the manufacturer’s instructions.