Cy3 conjugated goat α rabbit antibodies

Transfected S2 cells were grown on cover slips in 12-well plates. At 48 h after transfection, cells were washed with PBS, fixed with 4% PFA in PBS at room temperature for 10 min, and blocked with 1% BSA in PBS for 30 min. Cells were incubated with primary polyclonal anti-HhN antiserum (rabbit IgG, Santa Cruz Biotechnology, United States) at 4°C for 12h, washed 3 times with PBS and incubated with secondary Cy3 donkey anti-rabbit IgG antibodies (1:300) (Dianova) for 2 h at room temperature. DAPI (1 μg/mL) was added to all incubations. Cells were washed 3 times with PBS and mounted in mounting medium (Vector Laboratories). Wing discs were fixed, permeabilized and stained with anti-β-galactosidase antibodies (Cappel, MP Biomedicals) and Cy3-conjugated goat-α-rabbit antibodies (Jackson Immuno Research). Posterior Hh-producing cells were detected with monoclonal antibodies directed against engrailed (4D9, DSHB) and Alexa488-conjugated donkey-α-mouse antibodies (Thermo Fisher). Images were taken on an LSM 700 Zeiss confocal microscope with ZEN software. Maximum intensity projections are shown and individual sections are shown where indicated. Orthogonal views were created using ImageJ software.

Wing discs were fixed, permeabilized and stained with anti-β-galactosidase antibodies (Cappel, MP Biomedicals) and Cy3-conjugated goat-α-rabbit antibodies (Jackson Immuno Research). Posterior, Hh-producing cells were detected with monoclonal antibodies directed against engrailed (en 4D9, DSHB) and Alexa488-conjugated donkey-α-mouse antibodies (Thermo Fisher). Images were taken on a LSM 700 Zeiss confocal microscope using ZEN software. Maximum intensity projections are shown.