Protocol for induction and study of DNA double-strand breaks in mammalian cells using PALM microdissection and expansion microscopy

We present a protocol to induce localized DNA double-strand breaks (DSBs) in mammalian monolayer cells, using the PALM MicroBeam microdissector, followed by high-resolution visualization through expansion microscopy (ExM). We describe steps for laser microirradiation, immunostaining, and gel embedding. The protocol enables 4.5-fold physical expansion and confocal imaging of DNA damage sites. We quantify nuclear and damage site expansion using Fiji by measuring nuclear area and cross-sectional fluorescence profiles.