Alexa fluor 647 affinipure donkey anti rabbit igg h l

KYSE150 STAT3-KO cells were seeded onto coverslips for 24 h. Cells were transfected with Flag-STAT3α, STAT3β-HA, or a combination of both. After transfection, cells were fixed with 4% paraformaldehyde for 10–15 min, and then washed three times with PBS. Then, 0.1% Triton X-100 permeabilization reagents was added for 10 min. Cells were washed and blocked in 5% normal donkey serum for 1 h. Then, cells were incubated with anti-Flag M2 mouse monoclonal antibody (F3165, Sigma) and anti-HA (sc-7392, Santa cruz) overnight, and cells were stained with Alexa Fluor 488-conjugated donkey anti-mouse IgG (H + L) (1:500, 715-545-150, Jackson) and Alexa Fluor^® 647 AffiniPure donkey anti-rabbit IgG (H + L) (1:500, 711-605-152, Jackson) secondary antibodies for 60 min at RT, then washed three times with PBS and counterstained with DAPI (#D9564-10MG, Sigma) at RT for 10 min to visualize the nuclei. Images were obtained and processed by laser-scanning confocal microscopy (LSM800, Carl Zeiss).

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.