Leica confocal software lcs

For immunofluorescence staining, cells plated on coverslips were fixed in PBS 4% paraformaldehyde (PFA) at room temperature, blocked in PBS-1% bovine serum albumin (BSA), and permeabilized in PBS 0.2% Triton X-100. Stains were performed with primary antibodies α-tubulin-fluorescein isothiocyanate (FITC) (clone DH1A, F2168, 1:150) and γ-tubulin (clone GTU-88, T6557, 1:100) (Sigma Aldrich) and cleaved caspase 3 (Asp175) (#9661, 1:300) and phospho- histone H3 (Ser10) (D7N8E #53348, 1:300) from Cell Signaling Technology (Danvers, MA, USA) and p53 (DO-1 sc-126, 1:200) from Santa Cruz Inc. (Santa Cruz, CA, USA). Then samples were washed in PBS and incubated with secondary antibodies (Alexa-Fluor 488- or 568-conjugated anti-mouse or anti-rabbit antibodies; Invitrogen) for 1 h at room temperature. TO-PRO-3 iodide (Invitrogen, Carlsbad, CA, USA) were used to visualize nuclei and Alexa-Fluor 647-Phalloidin (Invitrogen) for F-actin staining. Coverslips were analyzed using the TCS-SP8 Confocal Systems (Leica Microsystems Heidelberg GmbH, Wetzlar, Germany) interfaced with the Leica Confocal Software (LCS) (version 3.5.5.19976, Wetzlar, Germany) or the Leica Application Suite (LAS) software (version 6.1.1, Wetzlar, Germany). At least 10 fields were scored for each cell population and experimental condition.

Mesothelial cells (derived from pleural lavage) were seeded on glass coverslips and allowed to grow until they reached complete confluence. 1.5 × 10⁵ EOC cells detached with 5 mM EDTA, labeled with vital green fluorescent lipophilic tracer DiO (Invitrogen) and washed with PBS were then plated on mesothelial layer in serum free medium for 24 hours.
For immunofluorescence analyses cells plated on coverslips were fixed in PBS-4% paraformaldehyde (PFA) at room temperature (RT), blocked in PBS-1% bovine serum albumin (BSA) and stained as indicated with primary antibodies: anti-α-tubulin-fluorescein isothiocyanate (FITC) (Sigma), phospho-Histone H3 (S10) (Upstate), ZO-1 (Cell Signalling) and γ-catenin (BD Biosciences). Then samples were washed in PBS and incubated with secondary antibodies (Alexa-Fluor 488-, 633- or 546-conjugated anti-mouse or anti-rabbit antibodies; Invitrogen) for 1 hour at RT. PI (5 μg/ml + RNaseA) or TO-PRO-3 iodide (Invitrogen) were used to visualize nuclei and Alexa-Fluor 647- or 546-Phalloidin (Invitrogen) for F-actin staining. Coverslips were mounted with glycerol/0.25% DABCO and analyzed using a the Leica Time Lapse AF6000LX workstation, the TCS-SP2 or the TCS-SP8 Confocal Systems (Leica Microsystems Heidelberg GmbH) interfaced with the Leica Confocal Software (LCS) or the Leica Application Suite (LAS) software.

Olympus FV1000 and Leica TCS SP2 microscopes were used for imaging with imaging and data analysis software packages FV10-ASW 2.0 Viewer and Leica Confocal Software LCS. Images were edited using Adobe Photoshop (Adobe Systems) and ImageJ software and assembled using Illustrator. All images are maximum intensity projections unless otherwise mentioned.