Gamma-H2AX immunofluorescence staining

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.