Donkey anti rabbit cy3 labeled antibodies

The ethanol-fixed cells were subjected to cyto-centrifugation followed by immunofluorescent staining to detect DNA damage-associated protein accumulation as microscopic foci at DNA double-strand break sites as described by Ahmed et al. [29 (link)]. Primary antibodies against γ-H2AX (Mouse anti-γ-H2AX; Merck) and 53BP1 (Rabbit anti-53BP1; Novus) were applied and detected with secondary goat anti-mouse Alexa-488 (Mobitec) and donkey anti-rabbit Cy3-labeled antibodies (Dianova). The number of radiation-induced DNA damage and repair protein foci was analysed by the same experienced investigator (HS) in 100 PBMC nuclei per sample by manual counting directly in a Zeiss Axioimager 2i fluorescence microscope of the ISIS fluorescence imaging system (MetaSystems) equipped with green and red double band pass filters (AHF Analysentechnik). Images were recorded at 630 × magnification with a Plan-Apochromat 63 × /1.40 oil lens.
To determine the number of radiation-induced foci per cell (RIF), the baseline focus values of each sample and time point (0-d, 0-4 h, 0-24 h) was determined. RIF are, for this study, defined as the difference between the number of foci per cell of the irradiated sample at the three time points 0 h, 4 h and 24 h (50 mGy-d, 50 mGy-4 h, 50 mGy-24 h) and the respective baseline value of the identical non-irradiated sample at the same time point.

Blood samples were taken from a healthy volunteer as indicated above. For analysis of DNA double-strand break foci we used the γ-H2AX + 53BP1 focus assay as previously described (Eberlein et al. 2015 (link); Lamkowski et al. 2014 (link)). Briefly, PBMC were isolated by Ficoll-paque (GE healthcare) density centrifugation at 1800×g for 20 min at room temperature. Cells were washed twice with PBS (pH 7,4; without Mg²⁺/Ca²⁺) and fixed in ice-cold 70% ethanol. For immunofluorescence staining (IF) we applied primary mouse anti-phospho(Ser139)-Histone H2A.X (Merck Chemicals; diluted 1:500) and rabbit anti-53BP1 (Novus Bio; diluted 1:500) antibodies and detected them with secondary goat anti-mouse Alexa-488 (Mobitec) and donkey anti-rabbit Cy3-labeled antibodies (Dianova) both at 1:1.000 dilution. Colocalization of γ-H2AX and 53BP1 foci was considered as indicative for DSB formation. Leukocyte nuclei (n = 100 per sample) were analyzed by an experienced investigator (H.S.) by manual focus enumeration using a Zeiss Axioimager 2i epifluorescence microscope equipped with a 63 × Planapochromat lens and red/green double bandpass filter (Chroma). Overlapping or deformed nuclei were excluded from the analysis. Only colocalizing γ-H2AX + 53BP1-positive foci were considered for enumeration. Images were recorded using the ISIS fluorescence imaging system (MetaSystems, Germany).