Anti rabbit igg conjugated to alexa 488

The fly intestines were dissected in PBS and fixed in PBS containing 4% formaldehyde for 20 min. After three 5-min rinses with PBT (PBS + 0.1% Triton X-100), the samples were blocked with PBT containing 5% normal goat serum and kept overnight at 4°C. Then the samples were incubated with primary antibody at room temperature for 2 hr and incubated with the fluorescence-conjugated secondary antibody for 2 hr at room temperature. Samples were mounted in the Vectashield mounting medium with DAPI (Vector Laboratories). We used the following antibodies: mouse anti-β-Gal (1:200; Clontech); mouse anti-Dl (1:50; DSHB); mouse anti-Pros (1:50; DSHB); nc82 (1:20; DSHB); anti-Slit (1:20; DSHB); guinea pig anti-Pros (1:3000, a gift from Tiffany Cook); rabbit anti-Robo2 (1:100, a gift from Barry Dickson); mouse anti-Robo1 (1:50; DSHB); mouse anti-Robo3 cytoplasmic (15H2) (1:50; DSHB); mouse anti-Robo3 extracellular (14C9) (1:50; DSHB); rabbit anti-CycT (1:1000; generated in Xinhua Lin's laboratory), and chicken anti-GFP (1:3,000; Abcam). Secondary antibodies used were goat anti-mouse, anti-chicken, anti-guinea pig, and anti-rabbit IgG conjugated to Alexa 488 or Alexa 568 (1:400; Molecular Probes). Images were captured with the Zeiss LSM 510 confocal system and processed with LSM Image Browser and Adobe Photoshop.