Alexa 488 conjugated goat anti mouse secondary antibody

The fixation and staining methods used for immunocytochemistry were performed as previously described (10 (link)). For immunostaining, AG1521 fibroblast cells were cultured on chamber slides and synchronized to the G0/G1 phases by contact inhibition. Following 30 min of treatment with monoglucosyl-rutin and radiation exposure at 37°C, the cells were fixed in 4% paraformaldehyde for 15 min, washed in PBS and then treated with 0.5% Triton X-100 for a further 10 min. Non-specific binding sites were inhibited using PBS with 10% goat serum at 4°C overnight. The chamber slides were then incubated with mouse monoclonal anti-γ-H2AX antibody (Millipore, Billerica, MA, USA) for 1 h, washed three times in PBS and incubated with Alexa 488 conjugated goat anti-mouse secondary antibody (Invitrogen Life Technologies) for 1 h at 37°C. The cells were washed four times in PBS and the nuclei were stained with Antifade Gold with DAPI solution (Invitrogen Life Technologies). Images of the cells were captured using a Z-stage motorized Zeiss Axioskop with Metamorph system (Carl Zeiss AG, Jena, Germany). The z-stacked images were stored and 50 cells from these were later scored in three independent experiments.

alx1;alx3/+ incross progeny were fixed at 2 dpf in 4% paraformaldehyde (PFA)/PBS overnight. Their trunk/tail portions were reserved for genotyping and the heads were exposed to 1% KOH/6% H₂O₂ to remove pigmentation and washed with PBST-X (1X PBS with 0.5% TritonX-100), followed by a 5-min proteinase-K (1:5000) treatment and 30-min 4% PFA/PBS post-fix at room temperature. Embryos were blocked with PBSTD-X (1X PBST, 1% DMSO, 10% BSA, 10% goat serum) for 2 h, at room temperature, incubated with mouse-anti-zn5 primary antibody (Zebrafish International Resource Center A28175, 1:100) and then Alexa-488-conjugated goat anti-mouse secondary antibody (Invitrogen AB_10013770, 1:500). Nuclei were labeled with DAPI (Molecular Probes; 1:5000). For imaging, embryos were mounted in VectaShield (Vector laboratories) and imaged on an Olympus IX81 inverted confocal microscope with the Fluoview 1000 confocal package, using a 60x water immersion objective (NA 1.10).

P16-P42 mice were perfused with 1 × PBS followed by 4% PFA. Brains were removed and stored in 4% PFA overnight and transferred to a 30% sucrose solution with 0.05% NaN3 prior to cryosectioning. Brains were cryosectioned in the sagittal or coronal plane through primary visual cortex at 50 μm thickness per section. Free-floating sections were washed three times with 1 × PBS prior to being placed in a 2 N HCl solution for 17 min for antigen retrieval of EphB4. They were subsequently washed three times in 1 × PBS and blocked with 15% Normal Goat Serum in 0.5% Triton for 1.5 h at room temperature. The sections were then placed in a solution containing a monoclonal mouse anti-EphB4 primary antibody (1:50, Invitrogen) with 0.5% Triton overnight on a shaker in 4 °C. The following day, slices were washed and incubated with an Alexa488-conjugated goat anti-mouse secondary antibody (1:1000, Invitrogen) for 2.5 h at room temperature on a shaker.