Anti phospho histone h3 ser 10 antibody

Monoclonal antibodies anti-PAX7 (1:10) and anti-MYOSIN HEAVY CHAIN (MHC - MF20) (1:20) were obtained from the Development Studies Hybridoma Bank (Iowa City, IA) (Cat. Pax7 and MF20). Mouse monoclonal anti-DESMIN (1:100) was obtained from Sigma (Cat. D1033). Mouse monoclonal anti-MYOD (clone 5.8 A, 1:50) was obtained from BD Biosciences (Cat. 554130). Anti-DYSTROPHIN antibody (1:200) was obtained from Abcam (ab15277). Anti-phospho HISTONE-H3 (Ser-10) antibody (1:100) was obtained from Cell Signaling (#9701). Anti-LAMININ DyLight 650 antibody (1:250) was obtained from Novus (Cat. NB300-144C). Anti-ß-Galactosidase (1:100) was obtained from Thermo Fisher (A-11132). Alexa Fluor 488-conjugated-Rat monoclonal anti-F4/80 (clone A3-1, 1:100) was from Biorad (Cat. MCA497A488T). Rabbit polyclonal antibody against Elmo was custom generated (Genscript), and the antibody recognized both ELMO1 and ELMO2. Alexa Fluor 488 chicken anti-mouse IgG, Alexa Fluor 488 chicken anti-rabbit IgG, Alexa Fluor 568 goat anti-mouse IgG and Alexa Fluor 568 anti-rabbit (1:1000) were obtained from Thermo Fisher.

A pool of 1h30-2h30 AEL embryos was fixed as for immunostaining except the fixation was in 1:1 heptane:1.8% formaldehyde/1X PBS (Thermo Scientific 28906) for exactly 10 min shaking at 450 rpm. Then embryos were rapidly quenched with 125 mM glycine PBS-1x and shaken for 1 min by hand. An anti-phospho-Histone H3 (Ser10) antibody (Cell Signalling #9701) was used at a dilution 1:200. Anti-mouse Alexa 488-conjugated (Life technologies, A21202) was used as a secondary antibody at a dilution 1:500. Embryos were kept in PBT until sorting.
Sorting was done using a COPAS SelectInstrument (Biometrica) with the following parameters: sorting limit low: 1, high: 256; PMT control: Green 650, Yellow 425, and Red 800. A restricted area of sorting (with the highest green signal) was selected representing ≈8% of the total population. A container was placed at the output of the non-selected embryos in order to re-pass them through the sorter to collect non-green embryos corresponding to interphase embryos. Right after the sorting, embryos were manually checked under a Leica Z16 APO macroscope by placing them on a glass cup and using Drummond Microcaps^® micropipettes to remove mis-sorted embryos individually. In all, 1000 embryos per tube were then dried by removing the PBT and kept at −80 °C.

GST tagged Ccnyl1 (77–367 aa) and 6×His tagged Ccnyl1 (77–367 aa) were expressed in E.Coli BL21 codon plus strain for anti-Ccnyl1 antibody generation and purification. Anti-Ccny antibody was as prepared previously described [10 (link)]. Other antibodies used were as follows: anti-β-catenin antibody (C-terminus) (BD, 610153); anti-β-catenin antibody (N-terminus) (Abclonal, A2064); anti-active β-catenin antibody (Millipore, 05–665); anti-Cre antibody (Novagen, 69050–3); anti-GAPDH antibody (Proteintech, 10494-1-AP); anti-Flag antibody (Sigma, F3165); anti-β-actin antibody (Sigma, 5316); anti-Lrp6 antibody (Cell Signaling Technology, 3395S); anti-phospho-Histone H3 Ser10 antibody (Cell Signaling Technology, 3377S); anti-phospho Lrp6 Ser1490 antibody for western blot (Cell Signaling Technology, 2568); anti-phospho Lrp6 Ser1490 antibody for immunofluorescence (gift from Christof Niehrs lab); anti-E2F1 IgG (Santa Cruz, sc-193).