Axioplan confocal microscope

Apoptosis was assessed in WT, p75^NTR−/− and p75^NTRCys259 CGNs treated for 24 h with either 500 μM or 1000 μM AraC starting at 4 DIV. Apoptotic cells were labelled using Click-iT plus TUNEL assay for in situ apoptosis detection kit (Thermo Scientific, Cat: C10617) according to manufacturer instructions. Neurons were also stained for cleaved caspase 3 (Cell Signalling Technology, 9761, 1:400), β-III tubulin and DAPI (Sigma; D9542; 1:10,000) following the protocol for immunocytochemistry explained below. For each experiment and treatment, neurons were cultured in duplicates, and at least 15 images were taken per coverslip with a Zeiss Axioplan confocal microscope. The number of cells positive for cleaved caspase 3 and TUNEL was quantified using NIH ImageJ software.

For the quantification and inmunodetection of specialized GFP tagged Pol κ and Pol η, 53BP1 and γH2AX respectively, cells were fixed in 2% paraformaldehyde (PFA)/2% sucrose and permeablized with 0.1% Triton X-100 in phosphate buffered saline (PBS) as described previously (Mansilla et al., 2013 (link)). For the detection of chromatin-bound protein ice cold, 0.5% Triton CSK buffer was used (10 mM Pipes, pH 7.5, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2). Proteins were preextracted 1 min or 5 min when detecting p21 or RPA/PCNA respectively. EdU was detected following manufacturer’s instructions (Click-iT EdU kit– C10338 Invitrogen). Blocking was performed overnight in PBS 2% donkey serum (Sigma, St. Louis, Missouri). Coverslips were incubated for 1 hr in primary antibodies: 53BP1(Santa Cruz, Dallas, Texas), γH2AX (EMD Millipore, Massachusetts), p21 (c-19 Santa Cruz), RPA (NA18 EMD Millipore), PCNA (Abcam), pH3 (ser10 Millipore). Secondary anti-mouse/rabbit-conjugated Cy2/Cy3 antibodies were from Jackson Immuno Research and anti-rabbit alexa 488 from Invitrogen. GFP-tagged specialized Y polymerases and GFP-PCNA were detected by GFP autofluorescence. Nuclei were stained with DAPI (SIGMA). Images were obtained with a Zeiss Axioplan confocal microscope or a Zeiss Axio Imager A2.

DNA fibers were analysed using a protocol previously used by us (Mansilla et al., 2013 (link)) with a minor change in the time of labelling. Exponentially growing cells were pulse labeled with CldU (20 µM) for 10 min, washed twice,and incubated with IdU(200 µM) for additional 30 min. Cells were trypsinized and lysed with 6 µl of 0.5% SDS, 200 mM Tris–HCL (pH 7.4) and 50 mM EDTA buffer onto clean glass slides, which were tilted, allowing DNA to unwind. Samples were fixed in 3:1 methanol/acetic acid and denatured with HCL (2.5 N) for 1 h, blocked in PBS 5%Bovine serum albumin (BSA) for 15 min and incubated with the mouse anti-BrdU (Becton Dickinson, USA) to detect IdU, donkey anti-mouse Cy3-conjugated secondary antibody (Jackson Immuno Research, West Grove, Pennsylvania), rat anti-BrdU (Accurate Chemicals, Westbury, New York) to detect CldU and donkey antirat Alexa 488 secondary antibody (Invitrogen). Slides were mounted with Mowiol 488 (Calbiochem), and DNA fibers were visualized using a Zeiss Axioplanconfocal microscope. Images were analysed using Zeiss LSM Image Browser software and Image J software. Each data set is derived from measurement of 85–100 fibers.