Fig. 2. Co-localization of RPA34 subunit with RPA70 and DNA replication foci. (A) Sperm nuclei were incubated in calcium-activated egg extracts and samples were fixed 30 and 60 minutes later and analyzed by fluorescence microscopy. DNA was detected using Hoechst (Aa,d). The RPA34 subunit was detected using the specific monoclonal antibody and a Texas Red-conjugated anti-mouse antibody (Ab,e). The RPA70 subunit was detected using the specific polyclonal E-Ky antibody (Materials and Methods) and a FITC-conjugated anti-rabbit antibody (Ac,f). (B) Nuclei were treated with 0.3% Triton X-100 before formaldehyde fixation at the indicated times, and the analysis was carried out using confocal microscopy. RPA34 (Ba-d) and RPA70 (Be-h) were visualized as in panel A. The overlap of RPA34 and RPA70 signals is shown (merge). Bar, 5 µM. (C) Sperm nuclei were incubated in egg extracts, and samples were pulse labeled for 3 minutes with biotinylated nucleotides. Three different nuclei are shown. RPA34 was detected using the monoclonal antibody and a secondary FITC-conjugated anti-mouse antibody. Nucleotide incorporation was visualized with Texas Red-conjugated streptavidin. Analysis was performed by deconvolution and the merge of the two signals is shown (merge, yellow). Bar, 5 µM.