Dylight 488 conjugated anti mouse antibody

Staged embryos were fixed overnight in 4% paraformaldehyde in 0.1M phosphate buffer with 0.15 mM CaCl2 (pH 7,3). After rinsing twice 0.1M phosphate buffer, embryos were permeabilized in acetone at −20C for 7 minutes. After several washes the embryos were incubated for one hour in block solution (0.1 M phosphate buffer, 2% bovine serum albumin, 1% dimethylsulfoxide [DMSO] and 0.5% Triton X-100 and 2% normal goat serum). The primary antibodies used were anti-laminin (rabbit 1:200, Sigma), anti-GFP (mouse 1:1000, Life Technologies) and anti-zebrafish Dlx3b (Mouse 1:250, ZIRC). After 3 washes during 2 hours the embryos were incubated in the secondary antibody, which were Dylight488 conjugated anti-mouse antibody (Goat 1:500, Jackson Immuno Research), Alexa568 conjugated anti-rabbit antibody (goat 1:500, Molecular Probes), and Cy5 anti-mouse antibody (goat 1:500, Jackson Immuno Research). Embryos were then rinsed in 0.1 M phosphate buffer and 1% DMSO 3 times for 1 hour and then stained for DAPI (1ug/mL, Sigma), and mounted in 1.5% low melting agarose (Sigma) in 0.1M phosphate buffer for imaging in Spinning disc confocal microscope (Olympus).