A1r storm confocal microscope

After the IR treatment, the cell cultures were incubated at 0.5 and for 1 h in their respective media at 37°C, with 5% CO₂. After incubation, the cultures were harvested, fixed with 2% paraformaldehyde for 5 min, and suspended in PBS. Individual cells were attached to the slide by centrifugation with CytoSpin Rotofix 32A (Hettich). The cells on the slide were immunostained with the γH2A.X (Abcam, ab81299) and pATM (Abcam, ab36810) antibodies. In brief, cells were blocked with blocking solution (5% SFB, 0.6% Triton X-100, PBS) for 1 h, washed with PBS, incubated with the antibodies overnight at 4°C, washed with PBS, incubated with secondary fluorescent antibody goat anti-rabbit IgG-FITC (Santa Cruz Biotechnology, sc-2012) or goat anti-mouse IgG-Fluorescein (Santa Cruz Biotechnology, sc-2010) for 2 h, washed with PBS, and mounted using Vectashield mounting medium with DAPI (Vector Laboratories, H-1200). The images were acquired with an Olympus IX71 inverted microscope or a Nikon A1R+STORM confocal microscope (Unidad de Microscopía at the Instituto de Investigaciones Biomédicas, UNAM). The images were analyzed with ImageJ and NIS-Elements Viewer software.

A total of 5 brains from the C57BL/6J strain and 5 brains from the C58/J strain were analyzed. The dorsal DG from each of the selected 4-section series per animal was imaged using confocal microscopy (Nikon A1R + STORM confocal microscope). We acquired 19–22 z-stack images (1.2 µm separation step between optical sections) at 60x magnification (WI objective, 2048 × 2048 pixels resolution). To recollect a representative sample of the whole DG from both groups, we imaged one field from the crest, two from the suprapyramidal blade, and one from the infrapyramidal blade.

Eight thousand cells per well were plated in Millicell EZ 4-well glass slides (Millipore, Burlington, MA, USA). Eighteen hours before treatments, the medium was changed for phenol red-free DMEM without fetal bovine serum and cells were incubated at 37 °C under a 95% air, 5% CO₂ atmosphere. After the treatments, cells were fixed for 20 min in 4% paraformaldehyde solution at 37 °C and permeabilized with 100% methanol for 6 min at −4 °C. Next, fixed cells were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature and incubated at 4 °C for 24 h with 1µg/mL of rabbit anti-pS400PR (ab60954, Abcam, Cambridge, UK) in 0.5% bovine serum albumin in PBS. The samples were rinsed thrice in PBS for 5 min each and incubated in the dark with 0.5 µg/mL anti-mouse Alexa Fluor 488-labeled secondary antibodies (A11034, Invitrogen, Carlsbad, CA, USA) for 45 min. Nuclei were stained with 1 µg/mL Hoechst 33342 solution (Thermo Scientific, Waltham, MA, USA). The cells were coverslipped with a fluorescence mounting medium (Biocare Medical, Pacheco, CA, USA). The samples were visualized in a Nikon A1R + STORM confocal microscope.
Specific characteristics of the antibodies described in Section 2.2 and Section 2.4 can be consulted in Table S1.