Fluorescein conjugated lectin concanavalin a

Animals were fixed as described above for FISH, bleached with methanol bleach solution, and permeabilized using with proteinase K (2 ug/ml) in PBSTx. Animals were blocked and incubated with primary antibody overnight, followed by incubation with goat anti-Rabbit IgG HRP Conjugate (Life Technologies) or goat anti-Mouse IgG HRP Conjugate (Life Technologies). Primary antibodies used were rabbit anti-phospho-Histone3[Ser10] (Millipore, clone 63–1C-8) 1:750, and mouse anti-BrdU (BD Biosciences) diluted 1:300. For BrdU labeling, samples were pretreated by incubation in 2N HCl (+0.5% Triton X-100) for 45 minutes followed by brief neutralization in 0.1M sodium borate. Signals were developed using Tyramide SuperBoost Kits (Invitrogen). Lectin stains were performed overnight at 4°C with fluorescein-conjugated lectin Concanavalin-A (Vector Labs). poly-ADP-ribose stains were performed on isolated cells as described previously (Tartier et al., 2003 (link)).