Vectorshield mounting medium

Heterogenous retinal cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 min, washed for 3 × 10 min of PBS, blocked in blocking solution (3% bovine serum albumin (g/mL), 0.1% Triton ×(−100) in PBS) for 20 min and incubated with mouse anti rat βIII-tubulin antibody diluted at 1:500 in antibody diluting buffer (ADB; 0.5% bovine serum albumin, 0.3% Tween-20 in PBS) for 1 h at room temperature. Cells were then washed for 3 × 10 min in PBS, incubated with the goat anti mouse AlexaFluor488 antibody diluted at 1:400 in ADB for 1 h at room temperature, washed for 3 × 10 min in PBS, mounted in Vectorshield mounting medium containing DAPI (Vector Laboratories, Peterborough, UK) and stored at 4 °C. Retinal cultures were imaged using a Leica DM6000B/AF6000 fluorescence imaging system (Leica, Wetzlar, Germany). The entire 8 well chamber was imaged and the number of βIII-tubulin⁺ RGC/RGC with neurites was quantified. Counts were conducted manually by an individual masked to the treatment groups. All cell cultures were performed both in triplicate, and three separate times (n = 3).

Animal tissue samples were embedded in paraffin and sectioned in 1µm slices. Immunostaining was performed after removal of paraffin as described for CaCo2 cells.
Cultured cell monolayers were prepared for immunostaining as described previously (27 (link)). In brief, CaCo2 cells were grown to confluence on coverslips. After incubation with or without different mediators, cells were fixated with 2% formaldehyde for 10 minutes and permeabilized with 0.1% Triton-X100 for 15 minutes afterwards, at room temperature. The coverslips were incubated at 4°C overnight using following primary antibodies at 1:100 in phosphate-buffered saline (PBS): rabbit anti-Desmoglein2 (MyBiosource, Kampenhout, Belgium); mouse anti-Claudin2 (ThermoFisher, Schwerte, Germany); mouse anti-PI-3-kinase (Santa Cruz, Heidelberg, Germany). As secondary antibodies, we used Cy3- or 488- labeled goat anti-mouse, goat anti-rabbit (all diluted 1:600, Dianova, Hamburg, Germany). Coverslips were mounted on glass slides with Vector Shield Mounting Medium as anti-fading compound, which included DAPI to visualize cell nuclei additionally (Vector Laboratories, Burlingham, CA). Representative experiments were documented with a confocal microscope (Leica LSM 780) (Zeiss, Oberkochen, Germany).

Cells were grown on polylysine-treated coverslips. For Leptomycin B treatment, 45 nM of Leptomycin B was added to cells for 30 min at 37 °C immediately prior to imaging. Cells were fixed in 4% paraformaldehyde and permeabilised in 1% Triton X-100. Cells were then blocked in 1% bovine serum albumin (BSA) made in PBS and incubated at 37 °C for 1 h. Primary antibodies were diluted 1:100 in 1% BSA and incubated with the cells for 1 h at 37 °C. Alexa Fluor 488 secondary antibody (Invitrogen) was diluted 1:500 in 1% BSA and incubated with the cells for 1 h at 37 °C. Coverslips were mounted in Vectorshield mounting medium (Vector Laboratories, Burlingame, CA, USA) and staining was visualized on an Olympus FluoView™ FV1000 confocal microscope.