Vectashield containing dapi

The evening prior to treatment, MOLM-14 cells were seeded at a density of 1×10⁵ cells/mL in RPMI 1640/10%FBS. The following morning, cells were treated with either DMSO, 7 at 1× or 6× IC₅₀, or 6.0 Gy gamma irradiation (followed by a one-hour incubation period). After 24 hours (or 1 hour for gamma-irradiated cells), cells were collected, washed with PBS containing 2% serum, and spun onto cytospin slides at a density of 1.5×10⁵ cells/slide. Slides were fixed for 15 minutes in 4% PFA followed by 3–15 minute washes in PBS. Cells were subsequently permeabilized with permeabilization buffer (50 mM NaCl, 3 mM MgCl₂, 10 mM HEPES, 200 mM sucrose and 0.5% Triton X-100 in PBS), and blocked overnight with 10% FBS in PBS containing 0.1% Triton X-100. Slides were subsequently washed, incubated with Gamma-H2Ax primary antibody (Millipore) and goat anti-mouse Dylight secondary antibody (Invitrogen) and mounted with Vectashield containing DAPI (VWR). After drying overnight, slides were sealed with nail polish and observed under a fluorescent scope.

Specimens at embryonic and postnatal stages were fixed in 4% paraformaldehyde for between 15 minutes and 3 hours, depending on the thickness of the tissue. Postnatal tissues were decalcified with 10% EDTA at pH 7.4 for 1–3 days at 4^o C. Embryonic and postnatal samples were equilibrated overnight with two changes of 30% sucrose/PBS at 4^oC. These samples were then embedded in O.C.T. compound (EMS) and sectioned at 10 μM in the sagittal plane. Frozen sections were washed with PBST (0.1% Tween 20) and blocked with 10% serum for 1 hour at room temperature, then incubated with the rabbit primary anti-BEK antibody (C-17) (sc-122, Santa Cruz, 1:200) and goat primary anti-GFP antibody (ab5450, Abcam, 1:500) overnight at 4^oC. The following day, sections were washed with PBST and incubated with Alexa Fluor secondary antibody at a 1:500 dilution in 10% serum for 1 hour at room temperature. Sections were then washed with PBST and mounted with Vectashield containing DAPI (VWR). Images were taken on the Leica TCS SP5/8 confocal microscope system.