Rat anti brdu

Antibodies used in this study included: UBR5 (#8755, CST^®, 1:1000); anti-mouse-BrdU (Becton Dickinson, different dilutions); anti-rat-BrdU (ab6326, Abcam; 1:500); PCNA-PC10 (Cancer Research, UK, 1:1000); Vimentin (V6389, Sigma-Aldrich, 1:1000); RNF8 (ab15850, Abcam; 1:2000); RNF168 (#ABE367, Millipore; 1:500); Tubulin (clone B-5-1-2, Sigma-Aldrich); Anti-Flag M2 (F3165, Sigma-Aldrich); 53BP1 (NB100-305, Novusbio, 1:2000); Chk1-PhosphoS345 (#2341 CST^®,1:1000); Chk1 (#2360 CST®,1:1000); Chk2-PhosphoT68 (#2661 CST^®, 1:500); Chk2(#2662 CST®, 1:1000); TRIP12 (ab86220, Abcam; 1:500); Histone H2A(ab18255, Abcam; 1:1000); polη (custom made, raised against the peptide: VQVEQRQNPHLRNKPC, 1:1000); polη-PhosphoS601(Eurogentec,(15 (link))); Histone H2A.X-PhosphoS139 (Upstate), RPA2 (1:1000, Millipore).

The immunofluorescence staining and cell quantification were carried out as described previously [2 (link),27 (link)]. Briefly, the cell samples were washed with PBS for 30 min followed by blocking with PBS (3% normal goat serum, 0.1% triton X-100 in PBS) for 1 h at room temperature. Primary antibodies were applied overnight at 4 °C. The following primary antibodies were used: anti-rat BrdU (Cat# ab6326, Abcam), anti-mouse GFAP (Cat# Z0334, DAKO, Santa Clara, CA, USA), anti-mouse Tuj1 (Cat# G712A, Promega), anti-rabbit Caspase 3 (Cat# AB3623, Millipore, Boston, MA, USA) and anti-mouse MAP2 (Cat# M9942, Sigma). The second day, fluorophore-conjugated secondary antibodies were applied for 1 h at room temperature. Images were captured and the numbers of BrdU⁺, Tuj1⁺, GFAP⁺ and Caspase3⁺ cells were quantified using ImageJ software (NIH).

BrdU incorporation was assessed as reported previously in Nagai et al. and Yamamoto et al.^{16 (link),20 (link)} On Day 6, BrdU (100 mg/kg; Sigma Aldrich) was administered intraperitoneally. Twenty-four hours after BrdU administration, mice were euthanized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde under anesthesia (n = 4). Brains were postfixed overnight in 4% paraformaldehyde at room temperature and dehydrated in 20% sucrose (4 °C for 1 day). Serial sections of the brains were cut (30-mm sections) on a freezing microtome. After the sections were washed with PBS containing Triton X-100 (PBST), they were incubated in 2 N HCl for 45 min and washed with PBST. After blocking in 3% normal donkey serum–PBST for 1 h, sections were incubated overnight with anti-rat BrdU (1:1,000; Abcam, Cambridge, United Kingdom) and DAPI (1:100,000; Thermo Fisher Scientific, Waltham, MA, USA) at room temperature. They were incubated with the secondary antibody (1:500, donkey anti-rat Alexa 594; Thermo Fisher Scientific) for 2 h. BrdU-positive cells were counted in the hippocampus using an Olympus microscope (Olympus, Tokyo, Japan).