Lsm 900 airy confocal laser scanner microscope

The mouse lung tissue was fixed and prepared as paraffin-embedded sections. The slides were deparaffinized and rehydrated, followed by antigen retrieval using citrate buffer in a microwave (650 W for 20 min). The slides were stained with primary antibodies EndoG (SAB3500213), γH2AX (JBW301, Merck), or p53BP1 (Cell Signaling), and DAPI (Life Technologies). Images were acquired by using a Zeiss LSM 900 Airy confocal laser scanner microscope (Zeiss, Göttingen, Germany). The 3D view of Z-stack images was measured in Imaris image analysis software (Bitplane AG, Zurich, Switzerland). The quantification of signal intensity was analyzed by ImageJ software. A minimum of 50 cells per condition were quantified.

The cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS. The cells were stained with the primary antibodies EndoG (sc-365359) and p53BP1 (Cell Signaling Technology, Inc., Beverly, MA, USA). After washing, the cells were incubated with Alexa 594 secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA). Images were acquired by using a 63x oil immersion objective on a Zeiss LSM 900 Airy confocal laser scanner microscope (Zeiss, Göttingen, Germany). A minimum of 50 cells were counted for each sample.