Lsm700 meta confocal laser scanning microscope

NAGLU +/+and NAGLU^−/− mice were anesthetized and transcardially perfused with saline solution containing 0.01 mL heparin, followed by 4% paraformaldehyde in 0.1 mol/L PBS saline solution. Brains were processed as previously described.⁷⁵ (link) Briefly, brains were rapidly removed on ice and postfixed overnight at + 4°C and cryoprotected in 30% sucrose in 0.1 M phosphate buffer (PB) with sodium azide 0.02% for 24 h at 4°C. Brains were sectioned frozen on a sliding cryostat at 40 μm thickness, in rostrum-caudal direction. Afterward, free floating serial sections were incubated with PBS Triton X-0.3% and blocking solution (0.5% milk, 10% FBS, 1% BSA) for 1 h and 30 min. The sections were incubated overnight at + 4°C with the primary antibody anti-COX1. The sections were then incubated with the corresponding florescent-labeled secondary antibodies, Alexa 488/Alexa 594 conjugated antimouse/antirabbit IgG. Nuclei were counterstained with Hoechst. Images were observed using a Zeiss LSM700 META/laser scanning confocal microscope (Zeiss, Oberkochen, Germany). Single images were taken with a resolution of 1024 × 1024.⁷⁶ (link)

For immunostaining, cells were seeded on sterile coverslips at 4 × 10⁵ cells in each 60 mm culture dishes, cultured for 48 hours, then irradiated and incubated (37°C, 5% CO₂) for indicated time. The irradiated cells were fixed with 4% paraformaldehyde for 10 min at room temperature and in pre-cooling methanol for 20 min at -20°C, permeabilized with 0.5% Triton X-100 in PBS for 10 min. Nonspecific binding sites were blocked with 5% nonfat-dried milk in PBS for 2 hours at room temperature before probing with primary antibodies. Anti-Ki67 rabbit polyclonal (ab15580, Abcam), anti-pATM rabbit polyclonal (ab81292, Abcam) and anti-53BP1 rabbit polyclonal (ab36823, Abcam) antibodies were used. Secondary antibodies (anti-mouse or anti-rabbit, Santa Cruz) conjugated with Alexa Fluor 488/594 were incubated for 1 hours. Images were obtained using a Zeiss LSM 700 Meta laser scanning confocal microscope (Zeiss). For counting foci, we used the spot detection function of the Imaris software. Quantification of foci was done from images of 100–120 cells for each time point from three independent experiments.

The transgenic GFP fusion lines were stained with 5 μg/mL propidium iodide (PI) for 10 s, and then washed with water. The confocal images were obtained with a Zeiss LSM-700 Meta confocal laser scanning microscope (CLSM) using 488-nm laser lines for GFP excitation. Image processing was performed with Photoshop version 7.0 (Adobe Systems, CA, USA).