Alexa 488 555 conjugated secondary antibody

Staining and visualization of DNA damage repair foci was essentially performed as described 52 (link). Cells were plated on chamber slides to achieve subconfluency for fixing. Cells were fixed with 4% PFA, permeabilized with 0.5% TritonX-100, and blocked with 5% goat serum in PBS 0.1% TritonX-100 (all Sigma-Aldrich). Cells were incubated with anti-γ-H2AX mouse monoclonal antibody (Millipore; at 1:500 dilution), anti-53BP1 (Abcam; 1:500), anti-PCNA rabbit polyclonal antibody (Abcam; 1:200), or anti-RAD51 mouse monoclonal antibody followed by incubation with rabbit or mouse Alexa-488/-555 conjugated secondary antibody (Invitrogen) at 1:1,000. All slides were counterstained with DAPI, examined and photographed by fluorescence microscopy (Olympus BX51).
Cryosections from human tissue samples were fixed with 2% PFA for 15 minutes and permeabilized with 0.5% TritonX for 10 minutes afterwards. Following 1 hour blocking at room temperature with 5% goat serum/PBS, slides were incubated overnight with primary antibody in a humid chamber at 4°C. Mouse anti-PCNA was co-incubated with rabbit polyclonal anti-53BP1. Secondary antibody including goat anti-rabbit IgG (Alexa Fluor488, Invitrogen, 1:1000 dilution) and goat anti-mouse IgG (Alexa Fluor555, Invitrogen, 1:1000) was incubated at room temperature for 1 hour. Slides were mounted in Fluoroshield with DAPI (Sigma, F6057).

First, deparaffinisation and antigen retrieval were performed on 6 µm back skin sections using the Trilogy pretreatment solution (Cell Marque). Then, the sections were incubated in 10% donkey serum (Abcam) diluted in PBS containing 0.3% Triton X-100, at room temperature for 1 hour. Subsequently, the sections were incubated with primary antibody against BrdU (rat, Abcam) or GFP (rabbit, Santa Cruz) at 4 °C overnight. After washing 3 times with PBS containing 0.1% Tween-20, the sections were incubated with the appropriate Alexa 488/555 conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. After washing 3 times with PBS containing 0.1% Tween-20, the samples were finally mounted in Vectorshield hardset mounting medium with DAPI (Vector Laboratories). Lgr5/6GFP and Krt14 expression in primary SCC cryosections was visualised by incubation with anti-GFP (chicken, Abcam) and anti-Krt14 (rabbit, Covance) antibodies, respectively.