Prolong gold antifade with dapi

DRG neurons were seeded on pre-coated coverslips in 24-well plates and processed as we described [31 (link),30 (link)]. Briefly, coverslips were washed 2x with PBS, fixed in 4% paraformaldehyde, rinsed, permeabilized with 0.1% Triton X-100/0.1% sodium citrate in PBS (9 min) and blocked in PBS with 3% BSA (w/v)/1% Donkey serum (v/v) for 40 min/37°C. Primary antibodies were: rabbit anti-Neurofilament 200 (1:20,000, Sigma-N4142), mouse anti Pol κ (1:12,000, Sigma-WH0051426M), mouse anti γH₂AX (1:4,000, Millipore #05-636). Coverslips were washed 3x in 1% BSA in PBS, incubated 45 min in the dark with 488 and 594 Alexa-dye conjugated anti-mouse and anti-rabbit IgG (Life Technologies). Coverslips were mounted with Prolong^® Gold Anti-fade with DAPI (Life Technologies) and viewed/captured with 40x objective using Olympus IX71 fitted with QIC-F-M-12-C cooled camera (QImaging, Surrey, BC) and QCapture Pro (QImaging) software. Fluorescence intensity was quantified using ImageJ (National Institutes of Health) software.

Immunoblot analysis was performed by using antibodies against porcine B-cell lymphoma (BCL)-2 (Cell Signaling Technology, Danvers, Massachusetts), BAX (Cell Signaling Technology), B-cell lymphoma–extra-large (BCL-XL) (Cell Signaling Technology), caspase-3 (Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase. Expression of apoptosis regulatory protein levels were normalized to both total protein levels and glyceraldehyde-3-phosphate dehydrogenase. TUNEL staining was performed by using 10-mm thick sections obtained from the peri-infarct zone fixed in 4% paraformaldehyde/phosphate-buffered saline for 20 min. Slides were permeabilized on ice with 0.1% Triton X-100 in 0.1% sodium citrate, and sections were labeled in the dark at 37°C for 60 min. Slides were rinsed with phosphate-buffered saline, and nuclei were labeled with ProLong Gold Antifade with DAPI (Life Technologies, Grand Island, New York). Images were acquired by using an Eclipse E800 fluorescence microscope (Nikon Corporation, Tokyo, Japan) and Openlab version 5 software (Perkin Elmer, Waltham, Massachusetts). TUNEL-positive cells were counted at 10× magnification by an investigator blinded to experimental group and are expressed as a percentage of all nuclei.

Telomere FISH was performed using a PNA probe (Panagene, Daejeon, Korea) as described (Batista et al., 2011 (link); Gu et al., 2012 (link)). Proliferating T cells were treated overnight with 0.2 µg/ml colcemid (Life Technologies), swollen in KCl buffer (12.3 mM HEPES, 0.53 mM EGTA, 64.4 mM KCl), fixed in methanol/acetic acid (3:1) and dropped onto glass slides. Metaphase spreads were rehydrated in PBS, fixed in 4% formaldehyde and dehydrated in an alcohol series. Slides were incubated with hybridization mixture (70% formamide, 10 mM NaHPO₄, pH 7.4, 10 mM NaCl, 20 mM Tris buffer, pH 7.5), placed on a heating block at 80°C for 5 min to denature chromosomal DNA and incubated for 30 min to 2 hrs at room temperature with the PNA probe (0.05 µg/ml). After washing, slides were mounted with ProLong Gold Antifade with DAPI (Life Technologies) and analyzed with a Leica microscope (Leica, Heidelberg, Germany). To visualize MRE11A binding, metaphase spreads were first incubated with anti-MRE11A (Cell Signaling Technology, 4847P; overnight, 4°C) and secondary Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) antibody (Life Technologies, A-11008) for 1 hr. After washing, slides were dehydrated in an alcohol series, hybridization mixture was applied and slides were placed on a heat block (80°C) for 5 min followed by 1 hr incubation as above.