Fig. 3. Antibodies against phospho-serine 10 of histone H3 stain S. pombe nuclei through the cell cycle, but staining is radically reduced in G1 cells and highest on mitotic centromeres. In each case, the top panel shows H3SP staining and the bottom DAPI/DIC. All panels use the same concentration of primary antibodies. For (A-C) Cy3 anti-rabbit antibodies were used at a dilution of 1:2000, for (D-H) FITC anti-rabbit antibodies were used at a dilution of 1:100. (A) Asynchronous wild-type cells. Cell 1 is in G2, cells 2 and 3 are in M, and cell 4 is in G1/S phase. Note the strong H3SP chromatin fluorescence in all cells except for 4. (B) H3SP staining is strong in cells arrested in early S phase. Cells were grown to early log phase in rich medium at 30°C before the addition of hydroxyurea. The chromatin of these early S phase cells had bright H3SP staining. (C) A mixed culture of cdc10.v50 and cdc7.A20 cells were grown to early log phase in rich medium at 25°C before the temperature was changed to 36°C for a further 6 hours. The H3SP staining of the single nuclei in the three cdc10.v50 arrested cells is much lower than that of the multi-nucleate, actively cycling cdc7.A20 mutant cell on the left. (D-E) Asynchronous wild-type cells stained by a less sensitive procedure to visualise H3SP than that used in A-C. Nuclear staining is only seen in the mitotic cells. (F) H3SP and Nuf2.GFP localisation. In each case the first (top) image is a merge of the Nuf2.GFP fluorescence (second panel) and H3SP staining (third panel). The fourth panel shows DAPI staining, while the fifth is stained with DAPI/DIC. In each case the pole proximal spot of H3SP associates with Nuf2.GFP kinetochore fluorescence. The two signals are not entirely overlapping as revealed by the presence of red, yellow and green sectors in many such pole associated spots - for example, in the middle cell.
