Anti-ROBO1 [IPI-ROBO1.81] in Immunohistochemistry (Mouse)

Thirteen-day old mouse embryos (E13) were fixed by immersion in 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.4 (PFA) overnight at 4 °C. Samples were cryoprotected in a solution of 10% sucrose in 0.12 M phosphate buffer (pH7.2), frozen in isopentane at 50 °C, and then cut at 20 μm with a cryostat (Leica). Immunohistochemistry was performed on cryostat sections after blocking in 0.2% gelatin in PBS containing 0.5% Triton X-100 (Sigma). Sections were then incubated overnight with the following primary antibodies: goat anti-Robo1 (1:500; R&D Systems, #AF1749) or IPI-ROBO1.81 followed by 2 h incubation in species-specific secondary antibodies directly conjugated to Cy-3 fluorophores (Jackson ImmunoResearch #705-165-152 and #711-165-152 147 for goat anti-Robo1 and IPI-ROBO1.81, respectively). Sections were imaged with a fluorescent microscope (DM6000, Leica) coupled to a CoolSnapHQ camera (Roper Scientific).