Rabbit anti phosphohistone h3 h3p

Embryos were fixed overnight in 4% PFA and washed with PBT0.2 (1X PBS with 0.2% Triton X-100). Blocking was completed for 30 minutes at RT in solution of PBT0.2 with 2% goat serum. Proliferating cells were labeled with rabbit anti-phosphohistone-H3 (H3P, Millipore, Billerica, MA) primary antibody at 1∶250 and incubated overnight. Embryos were rinsed thoroughly with PBT0.2 and then incubated for 2 hours at RT with secondary antibody Alexa Fluor 488 rabbit anti-mouse IgG (Molecular Probes, Grand Island, NY) at 1∶400 for visualization by fluorescence microscopy. The same procedure was used to analyze optic nerve with zn-5 primary antibody to label axons. Mouse-conjugated zn-5 (ZIRC 021009) was diluted at 1∶200 and embryos were incubated overnight. Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, Grand Island, NY) at 1∶400 was used as secondary antibody. Apoptotic cells were labeled with rabbit anti- active- caspase3 antibody (BD Pharmingen), and Alexa Fluor 568 (Molecular Probes, Grand Island, NY) goat anti-mouse IgG was used as secondary antibody

Immunostainings on whole animals or sections were carried out as previously described (Cebrià and Newmark, 2005 (link)). Antibodies used were: mouse anti-SYNAPSIN (3C11; 1:100; Developmental Studies Hybridoma Bank), rabbit anti-phospho-Histone H3 (H3P) (1:600; Millipore), mouse anti-ARRESTIN (VC-1; 1:15,000; kind gift from H. Orii, University of Hyogo, Japan), mouse anti-PHOSPHOTYROSINE (P-TYR-100; 1:500; Cell Signaling Technology) and mouse anti-TMUS-13 (MYHC; 1:500; kind gift from F. Cebrià; Cebrià et al., 1997 (link)). Secondary antibodies were Alexa Fluor 488 goat anti-mouse, Alexa Fluor 594 goat anti-mouse and Alexa Fluor 647 goat anti-rabbit (Thermo Fisher Scientific). Images were taken with a LSM700 (Zeiss) and processed with Fiji (Schindelin et al., 2012 (link)). H3P⁺ cells/mm² were automatically counted using Fiji (Schindelin et al., 2012 (link)).