Cells were seeded onto coverslips and then fixed with 4% paraformaldehyde for 15 minutes at room temperature (RT). Sections (thickness: 4 μm) of FFPE tumor tissues or an NSCLC TMA including 40 tumor samples (US Biomax, Inc.) were deparaffinized, rehydrated, and then subjected to antigen retrieval (citrate buffer, pH 6.0). All samples (fixed cells or antigen-retrieved FFPE tissues and TMA) were then permeabilized with 0.2% Triton X-100 at RT and then incubated with blocking buffer (5% normal serum in TBS containing 0.025% Triton X-100 [TBSTx]) for 1 hour at RT. Samples were incubated with the following primary antibodies: anti-Ki67 (1:1000), anti-RGS2 (1:200), or anti–phospho-histone H3 (1:500), diluted in TBSTx containing 1% BSA. After washing 3 times with TBSTx and incubating with the corresponding fluorochrome-conjugated (Alexa Fluor 488 or Alexa Fluor 594) secondary antibodies diluted in TBSTx containing 1% BSA (1:500), nuclei were counterstained with DAPI (50 ng/mL) and then coverslips were mounted with a mounting solution (Dako, Agilent). Fluorescence was observed under a confocal microscope.

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