Journal: STAR Protocols

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

doi: 10.1016/j.xpro.2025.103938

Figure Lengend Snippet: Standardization of the 355 nm laser energy of the PALM Microbeam to induce DNA double-strand breaks (DSBs) (A) Detection of laser-induced damage was achieved using the γ-H2AX and the 53BP1 protein. Laser power was varied from 35% to 45%, while maintaining a constant displacement speed of the stage of 10 and a focus of 5. These parameters are controlled directly in the PALM RoboSoftware. The laser output is shown. Blue represents DAPI, green γ-H2AX and red 53BP1. Scale 10 μm. (B) High-resolution images were captured using the LSM 710 Confocal Inverted Microscope to observe the distribution of proteins associated with the DDR and their co-localization. Scale bar 10 μm. (C) Utilizing the Z-stack function of the confocal microscope, a three-dimensional reconstruction of the cell was performed to examine the distribution of proteins across different planes and nuclear sites. A total of 11 optical sections were acquired, spanning 4.41 μm in depth with a step size of 400 nm. Interestingly, the marking of both proteins (γ-H2AX and 53BP1) is absent in a certain area of the nucleus, which could potentially be a region without histones, such as the nucleolus, or a highly compacted area like heterochromatin. Scale bar 5 μm .

Article Snippet: Microscope slides , Corning , Cat#2947BCN.

Techniques: Inverted Microscopy, Microscopy