Rabbit anti phospho histone h3 ser10 antibody

MOs and FITC-dextran (50 ng/embryo) as a tracer were injected into the dorsoanimal region of four-cell-stage embryos. Injected embryos were fixed at the late gastrula to early neurula stages (stages12.5–13) and immunostained using rabbit anti-phospho-Histone H3 (Ser10) antibody (Millipore) as primary antibody and Alexa Fluor 555-conjugated antibody (Molecular Probes) as secondary antibody, as described previously [11 (link)]. Nuclei were stained with DAPI. Confocal microscopic analyses were performed with LSM 710 (Zeiss). Five embryos from each experimental group were used for counting the number of nuclei and pH3-positive nuclei in more than two separate regions (a total area was more than 0.1 mm²) of MO-injected and FITC-positive regions of each embryo. For rescue experiments, embryos were co-injected with MOs and nemp1, ran, or globin (negative control) mRNA together with EGFP-HA mRNA (200 pg/embryo) as a tracer, fixed at stages12.5–13, stained with DAPI and anti-HA antibody. Nuclei were counted in EGFP-HA positive areas. The statistical significance (P-value) was calculated using Student's or Welch's t-test after comparison of the variances of a set of data by F-test.

For lipid droplet analysis of Drosophila intestines or fat bodies, infected flies were dissected, fixed in 4% formaldehyde, washed 3 times in PBS supplemented with 0.1% tween 20 (PBT), and stained with 1 μg/ml DAPI (Sigma) and 2 μg/ml Nile Red (Sigma). Human cells were incubated with the indicated supplements for 3 days, fixed with 4% paraformaldehyde, incubated with BODIPY 493/503 (Thermo Fisher Scientific) and DAPI for 30 mins, washed with PBS, and mounted on slides for confocal microscopy. Lipid droplet number and size were counted using the ImageJ particle analyzer. For phospho-Histone H3 (PH3⁺) staining, guts were incubated first with a polyclonal rabbit anti-phospho-Histone H3 (Ser10) antibody (EMD Millipore) diluted in a ratio of 1:500 in PBT supplemented with 2% BSA. Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) antibodies (Thermo Fisher Scientific) were used in a 1:200 ratio to visualize PH3⁺ cells. Samples were then mounted in Vectashield mounting media (Vector Lab Inc) and imaged using an LSM700 confocal microscope (Zeiss).

Rabbit anti-phospho-Histone H3 (Ser10) antibody (Merck Millipore) was used in 1:400 dilution using the antibody staining protocol from47 . Briefly, the embryos were fixed in 4% paraformaldehyde/PBS-TT (1xPBS, 0.2% Triton X100, 0.2% Tween 20), washed five times in PBS-TT, blocked in 20% heat inactivated sheep serum (Sigma)/1% BSA in PBS-TT, and incubated with the primary antibody overnight at 4 °C. After eight 15 min PBS-TT washes, the embryos were blocked again and incubated with the goat anti-rabbit Alexa568 (Life Technologies) at 1:1000 dilution overnight. DAPI was added at the final concentration of 300 nM together with the secondary antibody to counterstain chromatin. The samples were then washed again eight times 15 min with PBS-TT, embedded in Vectashield (Vectorlabs) and imaged under a Leica SP5X CLSM.

The tube staining method was performed on dissected gonads fixed in 3% paraformaldehyde and methanol (Chen and Arur, 2017 (link)). The samples are washed using 1X PBST (0.1% tween), blocked with 30% NGS and incubated with primary antibodies at 4°C overnight. Appropriate secondary antibodies were added and incubated at room temperature for 1–2 h followed by three washes with 1X PBST with DAPI included in the final wash and samples were mounted on a 3% agar pad with Vectashield mounting medium. The primary antibodies used in this study are: Rat monoclonal OLLAS epitope tag antibody (1:200, Novus Biologicals, Cat. No. NBP1-06713) and Rabbit anti-phospho-Histone H3 (Ser10) antibody (1:200, EMD Millipore Cat. No. 06–570). Secondary antibodies were goat-anti-rat Alexa Fluor 568 nm and goat-anti-rabbit Alexa Fluor 488 (1:1,000, Invitrogen). pH3 was used as a control of the staining protocol allowing us to identify mature oocytes.