Kinetics for γ-H2AX foci in macaque plucked hairs after total-body irradiation.

(A) Representatives images of plucked hairs obtained from the fur and the head (upper panel) and from eyebrows and whiskers obtained from the face (lower panels). Plucked eyebrow hairs and whiskers with morphologies similar to the ones shown in the lower panels were found suitable for γ-H2AX detection. The black squares in the lower panel indicate the region of interest for γ-H2AX microscopy. (B and C) Representative images of sampled hair bulb regions of eyebrow hairs and whiskers taken from NHPs at the indicated times after 8.5 Gy-TBI (B) and at 1 day after the indicated TBI doses (C) are shown. Images in the bottom row are enlargements of the regions inside the white squares in the upper images. The rightmost image in (C) is an enlargement of the apoptotic region in the adjacent image. Arrows denote apoptotic cells in the other images. Green, γ-H2AX; red, DNA stained with PI. (D) Kinetics for γ-H2AX foci loss in plucked hairs for pre-irradiation (Pre IR), sham-irradiation (Sham IR) and at time points after the indicated TBI dose. Images of the hair apex were taken using laser-scanning confocal microscopy and γ-H2AX foci were counted in 100–500 cells. fpc: foci per cell. (E) The incidence of γ-H2AX foci at 1 and 2 days post TBI obtained by counting foci. Error bars signify standard deviations (n≥3). fpc: foci per cell. Least squares regression analysis show a direct relationship between radiation doses and γ-H2AX signals (slopes = 0.260103 and 0.194473 and correlation coefficients r>0, r = 0.844 and 0.880 for 1 d and 2 d post-IR respectively (p<0.001)). (F) Incidence of γ-H2AX relative amounts at 1 and 2 days post TBI obtained by immunoblotting. Error bars signify standard deviations (n≥2). Relative amounts of γ-H2AX in each sample were determined by comparison with total H2AX.