Sc 10809

Mouse tissues were fixed with 4% PFA/PBS for 3 hours at 4°C and cryopreserved in 30% sucrose in PBS. Tissues were embedded in OCT and stored at −80°C until use. Cryostat sections were cut at 10 μm and adhered to glass slides. Sections were washed with PBS, then incubated with 0.1 N HCl for 30 minutes at 37°C. After brief washing with PBS, sections were incubated with 0.5% Triton X-100 in PBS for 15 minutes at room temperature, then incubated with blocking buffer (3% BSA in TBST) for 30 minutes at room temperature. Sections were incubated at 4°C overnight with antibodies to BrdU (347580, BD bioscience; 1/100), H3K79me2 (ab3594, Abcam; 1/2000), Histone H3 (sc-10809, Santa Cruz; 1/2000), phosphorylated histone H3 (06–570, Millipore; 1/500) and cleaved caspase-3 (9661, Cell Signaling; 1/200). Sections were washed three times with TBST for 10 minutes and then incubated with appropriate secondary antibodies conjugated with Alexa 488 or 546 (A11001, A21208, Invitrogen; 1/300) for 1 hour at room temperature. Nuclei were stained with DAPI. Fluorescence microscopy was performed on a BX51 microscope with DP30BW CCD camera (Olympus) using 10x and 20x objective lenses. Images were collected with DP controller software (Olympus).