ll other chemical reagents were purchased from Sigma-Aldrich Corp. dyes at low concentrations in the pipette solution, typically 0.1 mM and below, which minimized phototoxicity but still 20685848 provided satisfactory fluorescence intensities. Histology For a histological visualization, part of each freshly dissected tissue sample was fixed for 3 days at 4uC in a 4% paraformaldehyde solution in 0.01 M phosphate buffer, routinely embedded in paraffin, and sectioned in 45-mm thick slices employing a rotatory microtome Microm HM350S. Tissue sections were stuck on microscope slides by electrostatic attraction and dried up to 12 h at 50uC. Staining with Masson’s trichrome and hematoxylineosin was used to characterize LSCC. The preparations were examined with the fluorescent microscope AxioImage M1. Bright field images were taken using EC Plan-Neofluar 10x/0.3, Plan-Apochromat 20x/0.8, EC Plan-Neofluar 40x/0.75, or Plan-Apochromat 63x/ 1.40 OI lens and the high-resolution color camera AxioCam HRc. Fluorescence Imaging and Dye Transfer Studies Fluorescence signals were acquired using the Olympus IX81 microscope with UPlanSApo 20x/0.85 OI lens, Orca-R2 digital camera, fluorescence excitation system MT10, and XCELLENCE software. For dye transfer studies, a given dye was introduced into the cell-1 of a pair through a patch pipette in the whole-cell voltage-clamp mode. Typically, this resulted in the rapid loading of the cell-1, followed by dye transfer via the TT to the neighboring cell-2. A whole-cell recording in the dye recipient cell was established,1030 min after opening the patch in the cell-1. This allowed measurement of gT and avoided dye leakage into the pipette-2 during the measurements of dye permeability. Evaluation of GJ permeability to dyes from changes in fluorescence intensity in both cells was previously described elsewhere. In brief, the cell-to-cell flux of the dye in the absence of transjunctional voltage can be determined from the changes of dye concentration in the cell-2 over the time interval as follows: JT ~ vol2:DC2 Dt 1 Immunohistochemistry of Cells and Tissues Cell culture. Cells were grown in 24-well plates with glass coverslips on the bottom, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.2% Triton X-100/PBS for 13679187 3 min. Coverslips were incubated for 1 h with the following primary antibodies: mouse anti-a-tubulin, rabbit anti-Cx43, mouse anti-Cx43, rabbit anti-Cx26, rabbit anti-Cx30, then rinsed with 1% BSA/PBS and incubated with secondary goat anti-mouse IgG H&L or with donkey anti-rabbit IgG for 30 min. The F-actin network was visualized using Alexa 
Fluor 594 phalloidin; coverslips were incubated with the dye for 30 min at 37uC. Coverglasses were attached using Vectashield Mounting Medium with DAPI and sealed with clear nail polish. MitoTracker Green was used to stain mitochondria in live cells following the manufacturer’s 937039-45-7 web instructions. Analysis was performed using the Olympus IX81 microscope with UPlanSApo 20x/0.85 OI or PlanApo N 60x/ 1.42 OI lens and the Orca-R2 digital camera with the fluorescence excitation system MT10 and XCELLENCE software. Tissues. Freshly dissected tissues were immersed in 4% paraformaldehyde in PBS for 24 hours at 4uC, then transferred to 20% sucrose in PBS for 24 at 4uC, and frozen on specimen plates by using a TBS tissue freezing medium. Tissue samples were sectioned at a thickness of 25 mm in a microtome cryostat at 220uC. Sections were collected on SuperFrost slides and air