Embryos were fixed in 4% PFA overnight, embedded into OCT compound, and frozen. Frozen samples were sectioned at 5 μm thickness, dried up on slide glass, fixed in 4% PFA, and endogenous peroxidase activity was quenched by incubating the slides with 0.3% hydrogen peroxide. The sections were subjected to antigen retrieval by microwaving in sodium citrate (0.01 M, pH 6.0) as the antigen unmasking solution and then incubated overnight at 4 °C with the primary antibody. Antibodies used for this experiment are as follows: PCNA (PC 10, monoclonal, Sigma-Aldrich, St Louis, MO, USA), p27 (mouse monoclonal, Cell Signaling Technology, Danvers, MA, USA), activated caspase-3 (Cell Signaling Technology), BrdU (Bu20a, mouse monoclonal, Cell Signaling Technology), Nestin (clone 401, monoclonal, Merck Millipore), Hes5 (AB5708, polyclonal, Merck Millipore), and CDKN2A/p16INK4a (2D9A12, monoclonal, abcam).
Immunostaining with the mouse monoclonal antibody was carried out using an ABC kit (Vector Laboratories, Burlingame, CA, USA). After incubation with antibodies, sections were washed three times in PBS and then incubated with the appropriate biotinylated secondary antibody (DAKO and Vector Laboratories) followed by StreptABComplex/HRP (DAKO and Vector Laboratories) following the manufacturer’s instructions.
Immunostaining with the mouse monoclonal antibody was carried out using an ABC kit (Vector Laboratories, Burlingame, CA, USA). After incubation with antibodies, sections were washed three times in PBS and then incubated with the appropriate biotinylated secondary antibody (DAKO and Vector Laboratories) followed by StreptABComplex/HRP (DAKO and Vector Laboratories) following the manufacturer’s instructions.