4uC, washed in PBS, and then incubated with goat anti-rabbit and goat anti-mouse secondary antibodies, conjugated with AlexaFluor 555 and 488 (Invitrogen, Carlsbad, USA), respectively, at room temperature for 1.5 hours. Negative controls were done by omitting the primary antibodies, by incubation in rabbit serum, or by using inadequate primary antibodies. Sections were examined with a T300 (Nikon, Tokyo, Japan) fluorescence microscope. Immunolabelling for TEM Lowicrly embedded sections. Lowicryl embedded ultrathin sections from chemically fixed and high pressure frozen samples were processed according to the same protocol. Non-specific labelling was blocked by the PBS buffer containing 0.1% fish gelatine, 0.8% bovine serum albumin and 5% fetal calf serum (blocking buffer). Sections were incubated with polyclonal antibody made against highly purified bovine asymmetric unit membrane (anti-AUM antibody, a kind gift of prof. TT Sun, NYU), which reacts strongly with uroplakin IIIa, moderately with uroplakin Ia and Ib, and weakly with uroplakin II [10]. After rinse in washing buffer (blocking buffer without fetal calf serum), anti-AUM was detected with goat anti-rabbit IgG conjugated to 5 nm colloidal gold diluted 1:50 in blocking buffer. Sections were washed in washing buffer followed by de-ionized water. The 5 nm gold was silver enhanced with IntenSE (GE Healthcare, Amersham, UK). The enhancement times were 4 minutes. Negative controls were done by omitting the primary antibodies, by incubation in rabbit serum, or by using inadequate primary antibodies. Ultrathin cryo-sections. Ultrathin cryo-sections were prepared by modified Tokuyashu method. Briefly, urothelium, cut into ,1 mm3 pieces, was fixed in 4% paraformaldehyde plus 0.1% glutaraldehyde 0.1 M phosphate buffer for 2 hours, washed in PBS, embedded in 12% gelatine and cryoprotected by incubation in 2.3 M saharose. Samples were then stored in liquid nitrogen, and were subsequently cut with a Leica FCS cryo- ultramicrotom at 2120uC, and processed for immunolabelling with anti-Rab27b rabbit polyclonal antibody (SigmaAldrich, St. Louis, USA). Primary antibodies (diluted 1:10000) were incubated overnight at 4uC, washed in PBS, and incubated with goat anti rabbit secondary antibodies, conjugated with 10 nm colloidal gold (SigmaAldrich, diluted 1:40), at room temperature for 1.5 hours. Blocking non-specific labelling and negative controls were done as for Lowicryl embedded ultrathin sections. All immunolabelled sections were viewed in a Philips CM100 (Eindhoven, Netherlands) electron microscope, running at 80 kV. Freeze-fracturing Urothelium was fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, for 2 hours at 4uC. Tissue pieces were soaked in 30% glycerol in 0.1 M cacodylate buffer for 2 hours before freezing in liquid nitrogen. Replicas were produced in a Balzers BAF 301 freeze-fracture unit. Replica were cleaned by the technique of Van den Bosch and Jacob [46] and examined in the electron microscope. Electron tomography Semithin sections (200–300 nm thick) from high pressure frozen, Epon embedded tissues were cut perpendicular to the apical surface of the urothelium. ET was performed in a Tecnai-20 transmission electron microscope (FEI, Eindhoven, Netherlands) equipped with a tilting stage and running at 200 kV. A rotation holder (Gatan, Inc., Pleasanton, USA) was used in order to orient the sections. Series of tilted images (range: 265u to +65u) were acquired with a tilt increment of 1u by the help of the Xplore 3D software (FEI). This Electron Tomography of Fusiform Vesicles software requires a holder calibration procedure where the dislocation and the defocus of every tilt image are recorded. Before acquisition of a tilt series, an automatic adjustment of the eucentric height and an autofocus procedure were performed. During acquisition of tilted images dislocations caused by the mechanical imprecision of the stage are corrected. The volume of the semithin sections was reconstructed into serial slices using the software package Inspect 3D (FEI). This software implicates alignment and filter tools as well as a reconstruction module involving Fourier transforms and weighted back projection of the 2D tilt images. For studying whole 3D morphology and higher organization of FVs, ten serial semithin sections were cut and all sections were examined in order to find recognisable FVs that could be followed throughout their whole volume. For high resolution reconstruc- tions of individual FVs and of their higher organization, ET was done at magnifications 5000–96006. Modelling was done using IMOD software (http://bio3d.colorado.edu/imod/) [47]. Tomo- grams from each of the serial sections were joined along the Z-axis as described previously [48]. Supplement movies of the FVs models were compiled using MovieMaker 2.6 software (Microsoft, Redmond, USA) from sequences of individual JPEG files generated using the 3dmod viewer in IMOD. Quantitative analysis For quantitative analysis of mFVs orientation relative to the apical plasma membrane of umbrella cells, 5 contracted and 5 distended urinary bladders were used. Ultrathin sections (60 nm thick) were prepared from two Epon blocks of each animal and micrographs of 10 umbrella cells were taken at a magnification 210006 in a Philips CM100 microscope, running at 80 kv. Only umbrella cells with folded or flattened apical plasma membrane for contracted or distended urothelium, respectively, were analysed. In these cells, orientation and number of mFVs lying less than 1000 nm below the apical plasma membrane was determined. Orientation of FVs was determined as the angle of mFVs long-axis relative to the apical plasma membrane. Measurements were done on a personal computer using ImageJ ver. 1.44 software for Windows [49]. FVs, with their relative angles less than 45u were labelled as FVs parallel to the apical plasma membrane, while FVs, with their relative angles more than 45u were labelled as FVs perpendicular to the apical plasma membrane. Statistical analyses were performed with Microsoft Office Excel 2007 software. Statistical significance was tested with Student’s t-test. Supporting Information Movie S2 (WMV) Figure S1 Position of cytokeratin 20 network in the urothelial cells. Immunolabelling with anti-CK20 (green) antibody shows that cytokeratin 20 is distributed as a line (arrow) below the luminal membrane of umbrella cells (uc). Majority of FVs, immunolabelled with anti-AUM antibody (red), are posi- tioned in the central cytoplasm beneath the cytokeratin 20 location. Legend: blue – DAPI, ic - intermediate cell, bc – basal cell. Thickness of semithin cryo-section is 300 nm. Bar: 5 mm. (TIF) Movie S1 Tomogram of a single mature FV in the umbrella cell. (WMV) Single mature FV in the umbrella cell. Movie S3 Tomogram of stack of mature FVs in the central cytoplasm of umbrella cell. (WMV) PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e32935