Anti phospho histone h3 ser10 d2c8 ph3

Immunostaining was carried out as described previously [43] (link). The following antibodies were used: anti-synapsin (anti-SYNORF1, 1∶50; Developmental Studies Hybridoma Bank), anti-Smed-β-catenin2 (1∶1000; Chai et al., 2010) and anti-phospho-histone H3 (Ser10) (D2C8) (pH3) (1∶500; Cell Signaling Technology). Images were scanned, processed and quantified as described for FISH images. To avoid technical variance and obtain a reliable quantification of pH3+ cells, at least two independent experiments of RNAi and pH3 immunostaining were carried out for anterior amputation, incision, degrowth and growth experimental designs.

Immunostaining was carried out as described previously [18 (link)]. The following antibodies were used: anti-synapsin (anti-SYNORF1, 1 : 50; Developmental Studies Hybridoma Bank), anti-arrestin (VC1, a kind gift of Professor H. Orri), anti-phosphohistone H3 (Ser10) (D2C8) (pH3) (1 : 500; Cell Signaling Technology), and anti-α-tubulin (1 : 20, Developmental Studies Hybridoma Bank). Confocal laser scanning microscopy was performed using a Leica TCS 4D (Leica Lasertechnik, Heidelberg) adapted for an inverted microscope (Leitz DMIRB). Images were processed using ImageJ software. p-H3 positive cells were counted by visual inspection of confocal z-stacks of images corresponding to the whole animal. In the corresponding graphs error bars represent standard error of the mean.