Phospho histone h3 s10 antibody

Eyes were fixed in 4% PFA at 4 °C for 2 hr and washed in PBS. Retinas were dissected, permeabilized and blocked in PBS containing 1% BSA and 0.5% Triton at 4 °C overnight, washed in PBLec (1% Triton X-100, 1 mM CaCl₂, 1 mM MgCl₂, and 0.1 mM MnCl₂ in PBS), and incubated overnight in PBLec containing biotinylated isolectin B4 (1:25, Vector Labs). After three 20-minute washes in PBS, samples were incubated with FITC-streptavidin (1:100, Vector Labs) in PBS containing 0.5% BSA and 0.25% Triton X-100 at room temperature for 2 hr. Retinas were either flat mounted using Vectashield mounting medium (Vector Labs) or processed for labeling of mitotic cells using phospho-histone H3 S10 antibody (1:100, Cell Signaling 9701). The images have been taken in the same region of the retinas. The branching points, EC area, and mitotic cells per fields were quantified from multiple experiments as described previously (Gerhardt et al., 2003).

Mouse tissues were kept in 10% formalin neutral buffer (Sigma) at 4 °C. The specimens were then processed, embedded in paraffin, and sectioned at 5 μm. For immunostaining, heat-induced antigen retrieval was done using a pressure cooker (Retriever 2000, PickCell Laboratories). Sections were incubated with primary antibodies at 4 °C overnight. For immunofluorescence, primary antibodies were detected by DyLight 488 or Texas Red-conjugated secondary antibodies (1:200, Jackson ImmunoResearch Laboratories, Inc). Primary antibodies used were phospho-histone H3 S10 antibody (1:100, Cell Signaling 9701), VEGFR2 (1:200, Cell Signaling 2479), CD31 (1:50, Dianova DIA310), CD31 (1:50, Dako m0823), Quantification used integral optical density by Image-Pro Plus software. For immunohistochemistry, the samples were incubated with biotinylated goat anti-rabbit IgG followed by streptavidin-HRP (ImmunoCruz Staining System, Santa Cruz, CA), using DAB as a substrate.