Axiovert observer microscope z1

A total of 3 × 10⁴ NIH/3T3 cells were seeded per well in 24-well plates containing coverslips and cultured overnight. Cells were further incubated for 12 h in DMEM containing 0.5% FCS to induce ciliation. Cells were then treated with 1.5 μM purmorphamine and various concentrations of the test compounds or DMSO as a control. Twelve hours later cells were washed with PBS followed by fixation in 4% ice-cold paraformaldehyde for 10 min at room temperature. Permeabilization and blocking of non-specific binding was performed with a solution containing 0.1% Triton X-100 and 1% heat-inactivated horse serum in PBS for 30 min at room temperature. Samples were then incubated with mouse anti-N-acetylated tubulin antibody (dilution 1:5,000) and rabbit anti-Smo antibody (dilution 1:200) overnight at 4 °C followed by washing and subsequent incubation with Alexa Fluor 594-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated donkey anti-mouse antibodies (Invitrogen; 1:500 dilutions) and 4′,6-diamidino-2-phenylindole (0.1 μg ml⁻¹) for 45 min at room temperature. Coverslips were washed and mounted using Aqua Poly/mount (Polysciences. Inc USA). Images were acquired in a Axiovert Observer microscope Z1 (Carl Zeiss, Germany) using a Plan-Apochromat × 63/1.40 Oil DIC M27 objective.

Indirect immunofluorescence staining of cells has been described previously^{102 (link)}. Briefly, after reaching full confluency cells were permeabilized using Triton X-100 (Carl Roth GmbH, Karlsruhe, Germany) and fixed by paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA). Cells were stained by primary (1:50 anti-ZO1 rat, Santa Cruz Biotechnology, Dallas, USA, 1:800; anti-alphaTubulin rabbit, Sigma Aldrich, St. Louis, USA) and secondary antibodies (1:1000 Anti-rat IgG, Cy3, Jackson Lab. Inc., Waltham, USA; anti-rabbit IgG, Cy3; Jackson Lab. Inc., Waltham, USA) in PBS. DAPI (Sigma-Aldrich) was added in a dilution of 1:1000 to visualize DNA. Bright-field images were recorded using an Axio Vert Observer microscope Z1 (Zeiss).