Anti mouse igg atto647n

Anti-mouse immunoglobulin G (IgG)–Atto647N (Sigma-Aldrich, 50185) was used for labeling the mouse monoclonal anti-Dld antibody (Abcam, ab73331). For antibody conjugation, 1 μl of anti-Dld antibody (0.5 mg/ml) was mixed with 2.5 μl of anti-mouse IgG-Atto647N antibody (1 mg/ml) and incubated at room temperature for 30 min or on ice for 2 to 3 hours. After incubation, 2.5 μl of blocking buffer [mouse IgG (10 mg/ml) with 5 mM azide] and 0.5 μl of 0.5% phenol red (Sigma-Aldrich, P0290) were added for blocking the unconjugated anti-mouse IgG-Atto647N and vortexed thoroughly. Mixtures without anti-Dld antibody were used as control. Before microinjection, 24- to 26-hpf embryos were anesthetized in the Danieau medium supplemented with 0.003% tricaine followed by embedding in 1.2% low–melting point agarose. Ten nanoliters of labeled Dld antibody was injected into the hindbrain ventricle. The phenol red indicator serves to show the diffusion of antibody mixture into the forebrain ventricle. The injected embryos were then released from agarose and cultured in the Danieau medium for 2 hours before imaging.