Lsm 510 or 710

Drosophila immunofluorescence analyses were performed exactly as previously described [36 (link)]. For detection of mSmo in the primary cilium, Smo-/- cells stably transfected with vector control, mSmoWT or mSmoNQ4 plasmids were incubated overnight in culture media supplemented with 0.5% FCS plus SAG (100 nM). The following morning cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature followed by three 10 minute washes in 1xPBS. Fixed cells were permeabilized and blocked in PBGT (1xPBS+0.1% TritonX-100+5% Normal goat serum) for 1 hour, then incubated overnight with anti-Smo (SCBT) and anti- γ-tubulin (Sigma) antibodies. AlexaFluor 488 or 555 conjugated secondary antibodies (1:1000; Life technologies) were used. Slides were mounted using Vectashield with DAPI (Vector Labs). Immunofluorescence data were collected using Zeiss LSM 510 or 710 and images were processed using Zen2009 and Photoshop CS6. For all immunofluorescence experiments, multiple cells were examined over a minimum of three experiments, and representative images are shown. For quantification of ciliary localization, 96–126 cells were counted over three experiments and the percent of cells showing Smo in the primary cilium was determined. The paired t-test was used to determine statistical significance.

Free-floating sections were incubated (overnight, room temperature) with rabbit anti-Ki67 (1:1000, Leica, Cedarlane, Burlington, ON, Canada) or goat anti-doublecortin (DCX, 1:1000, Santa Cruz), then incubated in biotinylated species-specific secondary antibodies and the Avidin-Biotin complex (ABC kit, Vector laboratory). The immunoreactive material was detected in 3′-diaminobenzidine (DAB, brown precipitate). Immunoflurorescence staining with rabbit anti-calbindin (1:10000, Swant, Switzerland), anti-β-catenin (1:100, Santa Cruz), anti-Prox1 (1:3000, Millipore, Temecula, CA), or goat anti-DKK-1 (1:60, R&D system, Minneapolis) was detected with species-specific cyanin-3 (Cy3) or Alexa 594 (red) conjugated secondary antibody (Jackson Labs, West Grove, PA, USA). Sections (minimum 2-3/mouse) were observed under light microscopy or epifluorescence on a Leitz Aristoplan microscope (Leica, Montréal, QC, Canada), or confocal microscopy (LSM 510 or 710, Zeiss), digital pictures were taken and used for analysis. Staining specificity was confirmed by omitting primary antibodies.