Fig. 3.

Detection of γH2AX and TopBP1 foci in cells and normal tissue. Dermal cells from healer mice (MRL, Cong) and nonhealer mice (B6) were cultured on coverslips and then stained with antiphospho-histone H2AX). White bars are normal cells, black bars are irradiated cells (1 Gy). (A) The number of foci/cell by counting 20 nuclei/cell line at 60× is presented as three histograms (Aa) with MRL (Top); Congenic (Middle), and B6 (Bottom). (Ab) Percentage of γH2AX-positive cells determined by counting between 100–200 cells/treatment. (Ac) A representative γH2AX-stained MRL cultured dermal cell nucleus with foci. (Ba–d) Tissue sections from ear and small intestine of normal B6 and MRL mice treated with γH2AX antibody and DAB stains hair follicles, dermal and basal epidermal cells in the ear, and villous epidermal cells in the small intestine. (Ca) Western analysis of ear-derived dermal cells and (Cb) ear tissue is shown using chromatin-enriched tissue run on a 14% SDS/PAGE gel. (Ca) Loading controls for cells (lanes 1, 2) stained with Coomassie blue; γH2AX bands (lanes 3, 4) seen at approximately 15 kDa. (Cb) Loading controls for tissue stained with Coomassie blue (lanes 1–3); γH2AX bands (lanes 4–6). (D a and b) A similar analysis of TopBP1 staining of nuclear foci. (Dc) TopBP1 foci are seen in an MRL dermal cell nucleus.