Ph3s10

Cells grown on coverslips were fixed in 4% (w/v) paraformaldehyde and permeabilized in 0.5% (w/v) Triton X-100 in PBS for 30 min at room temperature (RT). After permeabilization, the cells were blocked with 3% (w/v) BSA for 30 min. Subsequently, they were incubated in primary antibody for 1 hr at RT. Antibody dilutions were 1:500 for anti-Oct4 (Santa Cruz, Dallas, Texas, sc-5279), 1:200 for anti-p-Oct4(S229), 1:200 for pH3S10 (Millipore, 05–598). Secondary antibodies used in immunostaining were Alexa Fluor 488, 568 (Invitrogen, Carlsbad, California).
Confocal micro-images were obtained by a confocal laser scanning microscope (Carl Zeiss, Germany, LSM 510 META).

The primary antibodies and their dilutions are: acetylated histone H3 at lysine 14 (acH3K14; 1:1,000; Millipore), acetylated histone H2B (acH2B; 1:2,000; Millipore), acetylated histone H4 (acH4; 1:1,000; Millipore), phosphorylated histone H3 at serine 10 (pH3S10; 1:1,000; Millipore), tri-methylated histone H3 at lysine 27 (3meH3K27; 1:2,000; Millipore), total H3 (1:2,000; Millipore), phospho-p44/42 MAPK (1:2,000; Cell Signaling Technology, Leiden, Netherlands) and p44/42 MAPK (1:2,000; Cell Signaling Technology). The secondary antibodies were goat anti-rabbit (1:25,000; LI-COR Biosciences) and donkey anti-mouse (1:20,000; LI-COR Biosciences).

Adult hermaphrodites raised at 20°C or 25°C were dissected in M9 buffer and flash frozen on dry ice before fixation for 1 min in methanol at -20°C. After washing in PBS supplemented with 0.1% Tween-20 (PBST), primary antibody diluted in in PBST was used to immunostain overnight at 4 °C in a humid chamber. Primaries used were 1:500 pH3S10 (Millipore, 06570), 1:4000 pH3T3 (Cell Signaling, D5G1I, Rabbit) 1:50 GSP-2 antibody (Colaiacovo lab), 1:300 LAB-1 antibody (Colaiacovo lab), 1:200 HIM-8 antibody raised in guinea pig (Dernburg lab), 1:200 SYP-1 antibody raised in goat, 1:500 H3K9me3 (Abcam ab8898), 1:500 H3K9me2 (Milipore Upstate 07–441), 1:500 H3K4me3 (Active Motif 39159). Secondary antibody staining was performed by using an Cy3 donkey anti-mouse or Cy-5 donkey anti-rabbit overnight at 4°C. All images were obtained using a LSM 710 laser scanning confocal and were taken using same settings as control samples. Images processed using ImageJ and Icy (http://icy.bioimageanalysis.org/). Intensity quantification was done by measuring total fluorescence in individual condensed chromosomes and subtracting the background levels obtained from mitotic nuclei as nucleoplasm levels varied greatly. Histone methylation intensity measurements were measured without background subtraction since only very few background was present.

Immunostaining was performed on a Bond Rx (Leica) or Ventana Discovery ULTRA (Roche) autostainer as per manufacturer’s instructions as previously described [40 (link), 41 (link)]. Primary antibodies and dilutions used in this study were as follows: pH3-S10 (Millipore; 06–570, 1:250), E2f3a (Millipore; 05–551, 1:100) and Myc-tag (Cell Signaling Technology; 2278, 1:100). EdU staining was performed following the manufacturer’s protocol (Life Technologies; C10337).