Rabbit gfp

Third-instar larval eye-antennal discs were dissected in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 30 min, washed in PBS + 0.1 or 0.3% Triton X-100 (PBT), and blocked in PBT + 1% bovine serum albumin (PBT/BSA). The tissues were incubated with the primary antibody in PBT/BSA overnight at 4°C. After washing off the primary antibodies, the tissues were incubated with the secondary antibodies in PBT for 1 hour at room temperature. After washing off the secondary antibodies, the samples were mounted in 80% glycerol or Vectashield (Vector Laboratories). For all eye imaginal disc experiments, we repeated the experiment at least twice with two independent biological samples from each genotype including the control genotype.
Antibodies used were as follows: mouse βGal (Sigma, 1:500), rabbit GFP (Invitrogen A11122, 1:500), mouse GFP (Invitrogen A11120, 1:500), rabbit Lgl (J. Knoblich, 1:500), mouse HA [Developmental Studies Hybridoma Bank (DSHB), 1:100], mouse N-intra (DSHB, 1:100), guinea pig Vha44 (M. Simmons, 1:100), rabbit Vap33 (H. Bellen, 1:1000), mouse Crb (DSHB, 1:50), and rabbit Patj (M. Bhat, 1:500). Secondary antibodies were as follows: anti-mouse Alexa 488, 568, 633, and 647; anti-rabbit Alexa 488, 568, 633, and 647; and anti– guinea pig Alexa 568. All secondary antibodies were used at a dilution of 1:500. DNA was stained with 1 µM DAPI.