Rabbit anti phospho histone h3 ser10

For immunocytochemistry, embryos were dissected and fixed in 4% paraformaldehyde-PEM (100 mM Pipes, 2 mM EGTA, 1 mM MgSO4), permeabilized in PBST (PBS, 0,1% Triton) and incubated overnight at 4°C with the primary antibody diluted in PBSTBN (PBST, 0,1% BSA, 5% NGS). The following primary antibodies were used at indicated dilution: mouse anti- engrailed/invected 4D9 (Abcam, 1:1), rabbit anti-phospho-Histone H3 (Ser10) (Cell Signalling, 1:1000) and rabbit anti-cleaved dcp-1 (Cell Signalling, 1:100). They were then washed with PBST and incubated with the secondary antibody: anti-mouse IgG-peroxidase (Merck, 1:100) or anti-rabbit IgG-peroxidase (Merck, 1:50) diluted in PBST for to 2 h and washed with PBST. The DAB reaction was performed for 3–5 min and embryos mounted for visualization in 50% glycerol in PBS. For fluorescent imaging, dissected ovaries were dissected in Ringer and fixed in 4% paraformaldehyde in PBS. Permeabilized in PBST and incubated for 20 min in a 300 ng/ml solution of phalloidin-TRITC in PBS. Rinsed with PBST and mounted in Vectashield medium with DAPI (Vector Laboratories, H1200). All samples were examined with a Zeiss Axiophot microscope, and images were subsequently processed using Adobe photoshop.

For Western blot analysis, 10 μg of protein were loaded on NuPAGE Novex 4–12% bis-tris Gels (Invitrogen) and processed as described (32 (link)). The following antibodies were used: rabbit anti-FGFR1 (1:500; no. 9740, Cell Signaling Technology), goat anti-OSMRβ (1:500; AF662, R&D Systems), mouse anti-myotrophin (1:500; 611830, BD), rabbit anti-GAPDH (1:5000; no. 2118, Cell Signaling Technology), rabbit anti-pERK (1:500; no. 9101, Cell Signaling Technology), mouse anti-panERK (1:500; 612641, BD), rabbit anti-ACTN1 (1:1000; ab68194, Abcam), rabbit anti-ACTN4 (1:1000; ab108198, Abcam), mouse anti-αSMA (1:1000; A5228, Sigma-Aldrich), rabbit anti–hexokinase I (1:1000; no. 2024, Cell Signaling Technology), mouse anti-MAD2 (1:1000; 610679, BD), mouse anti-Myh (1:1000; LS-B6307, LSBio), mouse anti-myomesin (1:100; clone M5, gift of H. M. Eppenberger), mouse anti-PCNA (1:1000; 555566, Bionity), rabbit anti–phospho–Histone H3 (Ser¹⁰) (1:1000; no. 9701, Cell Signaling Technology), rabbit anti–phospho-MEK1/2 (1:1000; no. 3958, Cell Signaling Technology), mouse anti–Ral A (1:5000; 610222, BD), rabbit anti-RUNX1 (1:1000; ab92336, Abcam), rabbit anti-SDHA (1:1000; no. 5839, Cell Signaling Technology), mouse anti-TIMP1 (1:1000; MAB9801, R&D Systems), and anti-goat Alexa Fluor 594 (1:1000; A-21468, Invitrogen).