Symmetric di methyl arginine motif sdma

After fixation or rehydratation, embryos were washed twice with Phosphate Buffered Saline/1% Triton X-100 (PBST), permeabilized with PBST/0.5% Trypsin for 30 sec and washed twice again with PBST. After blocking with PBST/10% Fetal Calf Serum (FCS)/1% bovine serum albumin (BSA) (hereafter termed ‘blocking solution’) for at least 1 h, embryos were incubated with antibodies directed against GFP (Torrey Pine, Biolabs), Prmt5 (Upstate #07405), MEP50/Wdr77 (Cell Signaling Technology #2823) or Symmetric Di-Methyl Arginine Motif (SDMA, Cell Signaling Technology #13222) in blocking solution overnight at 4°C followed by 5 washing steps with PBST. Embryos were then incubated with the appropriate Alexa Fluor-conjugated secondary antibodies (Molecular Probes) for at least 2 h at room temperature and washed three times. Nuclei were then stained with TO-PRO3 (Molecular Probes) and washed twice with PBST. Embryos were dissected, flat-mounted in glycerol and images were recorded on a confocal microscope as above. In situ hybridization was carried out as previously described [35 (link)]. Riboprobes were produced by linearizing PGEMT-cdh5 and PGEMT-fli1a vector by SacII and in vitro transcription by SP6 polymerase. Embryos were dissected, flat-mounted in glycerol and images were recorded on a confocal microscope as above or using the confocal Zeiss LSM 880.