Immunofluorescence Staining of Irradiated 3D Cultures

Cells were fixed with 4% paraformaldehyde at 4 h or 24 h after gamma exposure. Aggregated budding structures from 3D Matrigel cultures were processed and stained as previously described21 (link). Primary antibodies used for immunofluorescence include: cleaved caspase-3 (Cell Signaling #9664), anti-phospho-histone γH2A.X (1:400) (Millipore #DAM1479572). Proliferating cells were marked using the Click-iT Edu Alexa Fluor 488 kit according to manufacturer’s instructions (Invitrogen). Sections were mounted using Prolong Gold Antifade Reagent with DAPI (Life Technologies #P36931) and imaged using a confocal microscope. Each staining was performed on multiple sections in triplicate.