Alexa488 conjugated avidin

Antibodies against β-Actin (AC026, 1:10000), NUDT1 (A13330, 1:1000 for immunoblots, 1:200 for immunoprecipitation), NOX4 (A11274, 1:1000), H3 (A2348, 1:1000), NCL (A5904, 1:1000) were obtained from ABclonal. PLK1 (4513 S, 1:1000), Phospho-PLK1 (Thr210) (5472 T, 1:1000), MYC (13987, 1:1000 for immunoblots), cleaved-Caspase 3 (Asp175) (9661 S, 1:1000 for immunoblots, 1:100 for IHC) were obtained from Cell Signaling technology. Flag-tag (F1804, 1 μg for immunoprecipitation) was obtained from Sigma–Aldrich. N-MYC (sc-53993, 1:1000 for immunoblots, 4 μg for ChIP) and PCNA (sc-56, 1:2000 for IHC) were obtained from Santa Cruz Biotechnology. 8-oxo-dG (AB5830, 1:300 for IHC) and γH2AX (05-636, 1:200 for IHC, 1:100 for IF, 1:1000 for immunoblots) were obtained from Millipore. HRP-conjugated goat anti-mouse (115-035-003, 1:5000) and anti-rabbit (111-035-003, 1:5000) secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. The Phospho-NUDT1 S121 antibody was generated by Dia-An Technology by immunizing rabbits with phosphorylated S121 peptides (PDD-(phospho)S-YWF) conjugated with carrier protein keyhole limpet hemacyanin.
Other chemicals include 4-OH Tamoxifen, Tetracycline HCl, and TH287 (Selleck Chemicals); Acetylcysteine, BI6727, MG132, Pomalidomide and SBE-β-CD (MCE); CHX (Sigma–Aldrich); DAPI (Yeasen); Alexa488-conjugated avidin (Invitrogen).

Avidin binds to 8-oxo-dG with high specificity^{36 (link)} and was therefore used for 8-oxo-dG measurement. Cells were fixed on coverslip by methanol at −20 °C for 20 min and permeabilized with 0.1% Triton X-100 for 15 min at room temperature, before blocked by 5% bovine serum albumin, 0.1% Triton X-100 in TBS for 2 h at room temperature. Cells were then incubated with 10 μg/ml Alexa488-conjugated avidin (Invitrogen) in blocking solution for 2 h, then rinsed three times in TBS with 0.1% Triton X-100. DNA was finally counterstained with DAPI and slides were mounted and analyzed by confocal microscopy (Carl Zeiss).

A previously described assay based on the property of avidin to bind with high specificity to 8-oxo-dG was used for the 8-oxoG measurements [24 (link)]. Briefly, cells were fixed in methanol at −20 °C for 20 min and incubated for 15 min in TBS, 0.1 % Triton X-100. Blocking was performed in 15 % FBS, 0.1 % Triton X-100 in TBS for 2 h at room temperature (RT). Cells were then incubated with 10 μg/ml Alexa488-conjugated avidin (Invitrogen) in blocking solution for 1 h at 37 °C. Next they were rinsed twice in TBS, 0.1 % Triton X-100 for 5 min each round at room temperature. After a quick rinse in distilled water, DNA was counterstained with ToPro3-Iodide (LifeTechnologies) for 15 min at room temperature, followed by a final rinse in TBS.
Coverslips were mounted with ProLongGold (Invitrogen) and cells were observed under a Zeiss Axiolab fluorescence microscope equipped with a monochrome CCD camera. Analysis was conducted with NIH-imageJ, with respect to mean intensity in the nucleus (To-Pro3 served as a DNA reference). All experiments were repeated five times.

Slices containing biocytin-filled cells were fixed in 4% paraformaldehyde at 4 °C, then permeabilized and incubated with Alexa-488-conjugated avidin (Molecular Probes, Eugene, OR, USA). Slices were resectioned (70 μm) on a freezing microtome (Microm, Waltham, MA, USA) and mounted using Mowiol (Calbiochem, San Diego, CA, USA) mounting medium. Recorded cells were imaged with Nikon SD microscope at high magnification (40X objective). Three-dimensional reconstruction was performed for each cell using Neurolucida (MBF Bioscience, Williston VT, USA). The number of intersections at concentric circles (20 μm apart) was counted for each cell with the Scholl analysis module of Neurolucida (MBF Bioscience, Williston VT, USA) and analysed using an ANOVA.

SMCs were grown on glass coverslips and, at the time of harvest, were fixed with -20°C methanol and air-dried. Specimens were rehydrated in PBS and nonspecific binding was blocked with PBS containing 3% FBS. Samples were then incubated with 5 μg/ml biotinylated HA-binding protein (EMD Millipore, Billerica, MA) in PBS containing 3% FBS. After extensive washing with PBS containing 3% FBS, 4 μg/ml Alexa 488-conjugated avidin (Molecular Probes) was added. The samples were then extensively washed and mounted with mounting medium containing 4’,6 diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) to counterstain the nuclei. Samples were examined with a confocal microscope equipped for fluorescence. The HA-binding protein was omitted in assays serving as negative controls. Images were acquired using a Leica TCS-SP2 scanning confocal microscope equipped with four lasers, three PMT detectors and a spectrophotometer, operated with Leica Confocal Software and using an HC PL APO 20x/0.7 objective and an HCX PL APO 40x/1.25 objective (Leica Microsystems, GmbH, Wetzlar, Germany).