Deparaffinized sections were pretreated for antigen retrieval by boiling in the solutions: antigen retrieval solution, pH 9 (Nichirei Biosciences, Tokyo, Japan) for Ki-67, PSF1, PSF3, HIF-1α and NANOG; 10 mM citrate buffer, pH 6 for PSF2, SLD5 and MCM2; Immunosaver solution (Nissin EM, Tokyo, Japan) for CDC45. Endogenous peroxidase activity was blocked with peroxidase-blocking solution (Dako, Glostrup, Denmark). Sections were incubated overnight at 4 °C with rabbit monoclonal antibody against Ki-67 (1:1000; clone EPR3610; Epitomics, Burlingame, CA, USA), rat monoclonal antibody against PSF1 (1:300; clone aho57.2; GeneStem, Osaka, Japan), rabbit polyclonal antibody against PSF2 (1:3000; HPA057285, Atlas Antibodies, Stockholm, Sweden), mouse monoclonal antibody against PSF3 [1:500; clone aps3.2 (Nagahama et al.
2010a)], rabbit polyclonal antibody against SLD5 (1:1000; ab101346, Abcam, Cambridge, UK), rabbit monoclonal antibody against MCM2 (1:1000; clone D7G11, Cell Signaling Technology, Danvers, MA, USA) and rabbit monoclonal antibody against CDC45 (1:1000; clone EPR5759, Abcam), mouse monoclonal antibody against HIF-1α (1:20; clone 54/HIF-1α; BD Biosciences, Franklin Lakes, NJ, USA) and goat polyclonal antibody against NANOG (1:250; Novus Biologicals, Littleton, CO, USA). After the subsequent reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies (EnVision+, Dako, for mouse and rabbit primary antibodies; Simple stain, anti-rat, Nichirei Biosciences, for rat primary antibodies; ImmPRESS, anti-goat Ig, Vector Laboratories, Burlingame, CA, USA, for goat primary antibodies), color was developed with 3,3′-diaminobenzidine (DAB) and sections were counterstained with hematoxylin. For negative control experiments, primary antibody was replaced by non-immune immunoglobulin (Dako).