Cy3 sirna

For immunofluorescence analyses, antibodies specific for ZO-1, keratin 5, PKCζ, myosin II and phospho-myosin were purchased from BD Transduction Laboratories. Secondary antibodies included AffiniPure goat anti-mouse and goat anti-rabbit IgG Fab fragments from either Jackson ImmunoResearch Laboratories or from Molecular Probes. Localization and distribution of F-actin was followed by the staining of SMGs with Rhodamine- or Alexa-conjugated phalloidin^{3 (link),20 (link)}, while nuclei were visualized with monomeric cyanine nucleic acid stain, both from Molecular Probes. Evaluation of transfection efficiency was carried out with Cy3-siRNA, obtained from Dharmacon.

For functional perturbation of ZO-1 expression, siRNA was obtained from Dharmacon, while a non-targeting control was from Qiagen. Based on the initial determination of optimal inhibitory siRNA dose and time of treatment, concentration of 400 nM siRNA for 22–48 h was selected. The experimental design involved transfection of one gland from a pair of E12.5 or E13.5 SMGs with ZO-1 siRNA (S) while the second gland was designated for transfection with a non-silencing (NS) control using RNAiFect (Qiagen). To assure statistical significance, six SMG rudiments were cultured per filter for a total of three filters per condition, with each experiment being repeated independently at least three times. Transfection efficiency was assessed by tracking siRNA uptake by staining SMGs with Cy3-siRNA (Dharmacon). The effect of ZO-1 siRNA on the ZO-1 steady state transcript levels was measured using total RNA isolated from pooled SMGs from three independent experiments, according to standard procedures^{20 (link)}. ZO-1 transcript levels were determined by RT-PCR using 29S as a normalizing control^{20 (link)}.