Cy 3 affinipure donkey anti mouse igg h l

Cells cultured on confocal dishes were fixed with 4% paraformaldehyde (Sigma) for 15 min at room temperature, and permeabilized with 0.5% Triton X-100 for 20 min at 37 °C. After blocking with 5% BSA for 1 h at room temperature, cells were incubated with the primary antibodies against PAICS (Abclone, 1:100), γH2AX (CST, 1:200) and DAD51 (Abcam, 1:100) at 4 °C overnight. The next day, cells were incubated with corresponding secondary antibodies against Cy™3 AffiniPure Donkey Anti-Mouse IgG (H+L) or Alexa Fluor 647 AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson) at 37 °C for 30 min. 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was then applied to stain the nuclei. Finally, the images of immunofluorescence were obtained with the confocal laser-scanning microscope (Carl Zeiss, Jena, Germany).

Protein mixtures were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Carlsbad, CA), according to manufacturer’s instructions. Immunobloting analysis of FlaR expression was performed with rabbit anti-sera obtained against purified rFlaR and detected with Cy™3 AffiniPure Donkey Anti-Mouse IgG (H + L) (Jackson ImmunoResearch, West Grove, PA).

The cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Then they were washed three times with PBS 1x for 5 min. Next, the cells were permeabilized with 0.3% Triton diluted in 1x PBS for 15 min. Subsequently, blocking was done using a 5% BSA solution diluted in PBS 1x for 60 min. We then placed the primary antibody in question in PBS-BSA 3% at 1:100 (LMNA, cat. MA3-1,000, ThermoFisher scientific) for Lamin A/C; and 1:200 (Troponin T, cat. MS-295-P1, ThermoFisher scientific) for Troponin T, cardiac isoform, dilutions, and incubated overnight at 4°C. The following day, we washed 3x with 1x PBS for 5 min. After the washes, we placed the secondary antibody Cy™3 AffiniPure Donkey Anti-Mouse IgG (H + L) (cat. 715150, Jackson ImmunoResearch) diluted in PBS-BSA 3% at a 1:400 ratio for 2 h at room temperature. After incubation, we washed 3x with 1x PBS for 5 min. Finally, we placed DAPI (cat. D9542, Sigma-Aldrich) for 5 min and washed 3x times with PBS 1x for 5 min. The coverslips were sealed using 13 µL of Fluoroumont (ThermoFisher scientific). Images were taken with the ×100 objective on the Elyra PS.1 confocal microscope (Carl Zeiss), ZEN 2012 SP5 software (Carl Zeiss), at the Advanced Microscopy Unit (UMA) of the National Center for Structural Biology and Bioimaging (UFRJ, Rio de Janeiro).

Samples were incubated for 1 h with the following antibodies diluted in 5% BSA in PBS: rabbit polyclonal anti-53BP1 (ab36823, Abcam, Cambridge, UK), and mouse monoclonal IgG2b DynLL1/Pin1 (MAB2294, R&D systems, Minneapolis, MN, USA). Cells were washed 3 times in PBS and incubated for 45 min at room temperature with the following secondary antibodies: Alexa Fluor^® 647 AffiniPure Donkey Anti-Rabbit IgG (H + L) (711-605-152, Jackson-immunoresearch, West Grove, PA, USA) and Cy™3 AffiniPure Donkey Anti-Mouse IgG (H + L) (715-165-150, Jackson-immunoresearch, West Grove, PA, USA). Cells were then briefly re-fixed in 4% paraformaldehyde (wt/vol) and incubated overnight with the following primary antibodies: Alexa488-conjugated mouse anti phosphoH2A.X (ser39) (γH2A.X) (613406, Biolegend, San Diego, CA, USA) and rat monoclonal anti-Tubulin (MAB1864, Chemicon, Merck Life Science, Milan, Italy). Cells were then rinsed 3 times in PBS and incubated for 1 h with donkey anti-Rat IgG (H + L) biotin (a18749, ThermoFisher Scientific, Waltham, MA, USA) and for 45 min at room temperature with streptavidin Atto425 conjugated (S000-51, Rockland Immunochemicals, Pottstown, PA, USA). After washings, DNA was counterstained with Chromomycin, and finally, cells were briefly re-fixed in 4% paraformaldehyde (wt/v). Before image acquisition, PBS was replaced by STORM Imaging Buffer.