Alexa fluor 488 anti mouse igm secondary antibody

Cells were treated with compounds for 3 days, following which they were adhered to glass coverslips, washed with PBS and then fixed with 3% paraformaldehyde in PBS for 20 minutes. Fixed cells were rinsed with PBS and permeabilised with 0.5% Triton-X-100 for 5 minutes. PBS washed slides were incubated for 1 hour with 10 % FCS and 0.1% Triton-X-100 in PBS following which cells were stained with an anti-53BP1 monoclonal antibody (H-300, Santa Cruz, diluted 1:600), in combination with an 8-oxoguanine antibody (2Q2311, AbCam, diluted 1:400), where indicated, in 10 % FCS and 0.1% Triton-X-100 in PBS. After rinsing with PBS coverslips were incubated with an Alexa fluor® 568 goat anti-rabbit IgG secondary antibody in combination with an Alexa fluor® 488 anti-mouse IgM secondary antibody, where indicated, for 1 hour (Invitrogen, diluted 1:400) in 10 % FCS and 0.1% Triton-X-100 in PBS. After a PBS wash, DNA was counterstained with DAPI (Sigma-Aldrich) for 10 minutes and the coverslips were mounted in Fluorescent Mounting Medium (Dako). Images were analysed with a Zeiss fluorescent microscope at 63 times magnification with supporting software.