Alexaflour 568 conjugated goat anti rabbit

Tissue sectioms (5 μM thick) were deparaffinized and hydrated before the antigen retrieval step. Heat-induced antigen retrieval was performed at pH 6 for 20 min. The tissues were incubated with blocking buffer (4% BSA in 0.1% Tween-20+PBS) for 30 min. Sections were incubated with phospho-histone H2AX antibody (1:500, Cell Signaling, cat.#9718) in blocking buffer overnight at 4˚C. Following washes with PBST, tissue sections were incubated with Alexaflour 568 conjugated goat anti-rabbit (1:500, Invitrogen, A11011) in blocking buffer at room temperature for 1 hr. Sections were washed and mounted with Vectashield mounting medium with DAPI (Vector Laboratories) and images were taken with fluorescence microscope by using 40X objective.

Melanoma tissue sections were processed for TIF analysis by fluorescence in situ hybridization (FISH) as described previously (18 (link)). Tissue sections were hybridized with a FITC-conjugated telomere sequence (TTAGGG)₃-specific peptide nucleic acid (PNA) probe (PNA Bio, Thousand Oaks, CA) in formamide followed by incubation with a polyclonal anti-53BP1 antibody (1:500) (Novus Bio, Littleton, CO) and then with Alexaflour 568 conjugated goat anti Rabbit (1:500) (Invitrogen). Images were captured with a Deltavision microscope at 100x and then deconvoluted using Autoquant X3. TIFs were quantified using Imaris software.