Rabbit anti h3k9me3

The following primary antibodies diluted in PBST were used in these experiments: mouse anti-Dl, mouse anti-Arm (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), 1:200; mouse anti-GFP and rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), 1:1000; rat anti-GFP (Nacalai Tesque Inc., Kyoto. Japan), 1:1000; rabbit anti-γH2AvD (Rockland, Gilbertsville, PA, USA) 1:2000; rabbit anti-phospho-histone H3 (PH3, Millipore, Billerica, MA, USA), 1:1000; mouse anti-γ-tubulin (Sigma–Aldrich), 1:1000; rabbit anti-H3K9me3 (Millipore, Billerica, MA, USA), 1:200; and, mouse anti-HP1 (DSHB, Iowa City, IA, USA), 1:200. The following secondary antibodies diluted in PBST were used: goat anti-rabbit FITC (Jackson ImmunoResearch, West Grove, PA, USA), 1:400; goat anti-rabbit Cy3 (Jackson ImmunoResearch), 1:400; goat anti-mouse FITC (Jackson ImmunoResearch), 1:400; goat anti-mouse Cy3 (Jackson ImmunoResearch), 1:400; goat anti-rat FITC (Jackson ImmunoResearch), 1:400, goat anti-rabbit Alexa Fluor^® 647 (Jackson ImmunoResearch), 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes), 1:1000.

Zygotes were fixed in 3.7% paraformaldehyde (PFA) in PBS containing 0.2% Triton for 20 min. After 4x washes with PBS containing 10 mg/ml BSA (PBS/BSA), zygotes were treated with primary antibodies at 4°C overnight. The primary antibodies used in this study were mouse-anti-H3K27me3 (1/500, Active Motif, 61017), rabbit anti-H3K9me3 (1/500, Millipore, 07-442), and rabbit anti-FLAG (1/2000, Sigma-Aldrich, F7524). After 3x washes with PBS/BSA, samples were incubated with a 1:250 dilution of fluorescein isothiocyanate–conjugated anti-mouse IgG (Jackson Immuno-Research) or Alexa Flour 568 donkey anti-rabbit IgG (Life technologies) for 1 h. The zygotes were then mounted on a glass slide in Vectashield anti-bleaching solution with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Fluorescence was detected under a laser-scanning confocal microscope with a spinning disk (CSU-10, Yokogawa) and an EM-CCD camera (ImagEM, Hamamatsu) or Zeiss LSM800.
All images were acquired and analyzed using the Axiovision software (Carl Zeiss). The fluorescent signal intensity was quantified with the Axiovision software. Briefly, the signal intensity within the maternal pronuclei was determined, and the cytoplasmic signal was subtracted as background. Then, the averaged signal intensity of the no-injection control zygotes was set as 1.0.

Cell fixation and staining conditions were adapted from (48 ). Cells were permeabilized with Pipes–Hepes–EGTA–MgCl₂ (PHEM) buffer with 1% Triton X-100 and subsequently fixed in PHEM with 2% paraformaldehyde. Blocking was carried out in Tris-buffered saline with 10 mM EGTA and 2 mM MgCl₂ (TBSTEM) with 3% BSA. Cells were stained with rabbit anti-H3K9me3 (1:200, Millipore, cat: 07-442, lot: 2120113) and anti-H3K27me3 (1:200, Millipore, cat: 07-449, lot: 2275589) primary-antibodies. Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (1:200, Life Technologies, cat: A11008, lot: 1275894) was used for detection. Stained cells were mounted onto glass slides and imaged on the Leica AF6000 system with a HCX PL Fluortar 100.0 × 1.30 oil objective.