Zen lite 2012 blue edition

Coverslips had been fixed with 99% ethanol at −20 °C for 20 min and were subsequently air dried. Dried coverslips were stored on filter paper at −20 °C until further processing. Staining was performed as described previously [19 (link)] and included a blocking step in 10% (v/v) normal goat serum (NGS) and 0.1% (v/v) Triton X-100 in PBS. Afterwards, the coverslips were incubated for 2 h with primary antibodies (Table 2) followed by blocking in 0.2% bovine serum albumin in PBS (w/v) for 30 min. The corresponding secondary antibodies (Alexa–Fluor^®488 goat anti–mouse, Alexa–Fluor^®568 goat anti–mouse, Alexa–Fluor^®488 goat anti–rabbit, and Alexa–Fluor^®568 goat anti–rat; all from Thermo Fisher Scientific) were used in a 1:3000 dilution and cells were incubated for 60 min. Finally, coverslips were mounted onto microscope slides using DAPI–Fluoromount g (Southern Biotech, Birmingham, AL, USA). Analysis of Cx43 (abcam) and ZO–1 (Thermo Fisher Scientific, Dreieich, Germany) immunofluorescence was performed on images of PBS– (n = 2) and sucrose– (n = 3) treated cells. Using the square profile function of the software Zen lite 2012 blue edition (Carl Zeiss, Oberkochen, Germany), fluorescence maxima were determined on 3–5 square regions per image. Subsequently, the distance between fluorescence peak maxima was calculated according to Fischer et al. [25 (link)].