Alexa 647 conjugated goat anti mouse

For BrdU incorporation analysis, MEFs were pulse-labeled for 1 h with 100 μM BrdU (BD Pharmingen, 550891) before collection. Collected cells were fixed in –20 °C cold 70% ethanol and washed in blocking buffer (PBS-bovine serum albumin (BSA) 1%; BSA Sigma-Aldrich, A7906). For non-BrdU analysis, cells were permeabilized with PBS-1% BSA-0.5% Triton X-100, whereas, for BrdU analysis, cells were treated with 2 n HCl/0.5% Triton X-100 and neutralized with 0.1 m sodium tetraborate, pH8.5. Cell staining were performed using anti-BrdU (1:500; BD Pharmingen, 555627, clone 3D4), pS10-histone H3 (1:100; Cell Signaling Technology [CST] #9701) or pS139-histone H2AX (1:500; CST #9718) followed by their incubation with secondary antibody Alexa 647-conjugated goat anti-mouse (1:400; Invitrogen, A21235) or Alexa 647-conjugated donkey anti-rabbit (1:400; Invitrogen, A31573). Before analysis, cells were resuspended in PBS supplemented with 2.5 μg/ml propidium iodide (Merck, 537059) and 20 μg/ml RNase A (Sigma-Aldrich, R6513). Analysis of cells and BrdU incorporation was acquired on BD FACSCalibur or BD LSRII flow cytometers (BD Bioscience) and further analyzed using FlowJo 8.8.7 software.

The procedures were modified from the previous reports (Chen et al., 2021 (link)). Mice were anesthetized with 1% pentobarbital sodium and intracardially perfused with saline followed by 4% polyformaldehyde. The brains were removed, postfixed in 4% polyformaldehyde for 12 h, and then equilibrated in 30% sucrose. Coronal sections of 40-μm thickness containing the hippocampus were collected. Brain slices were blocked for 1 h with 5% bovine serum albumin and 1% Triton X-100 at room temperature and then incubated with primary antibodies (rabbit anti-NeuN 1:300, Cell Signaling Technology, Cat# 12943; mouse anti-GFAP 1:500, Cell Signaling Technology, Cat# 3670 and rabbit anti-Iba1 1:500, Cat# 01919741) overnight at 4°C. The next day, the slices were incubated with secondary antibodies (Alexa 488-conjugated goat anti-rabbit and Alexa 647-conjugated goat anti-mouse, 1:500, Invitrogen) for 2 h at room temperature after washing three times with PBS and then incubated with DAPI (Invitrogen) for 10 min and washed for another three times. The slices were mounted on microscope slides. Fluorescence images were captured using a confocal microscope (A1R, Nikon). Cell counting and fluorescent area calculation were performed by ImageJ.