Tcs sl confocal microscope

One out of four sections (3–6 per mouse) of the PRC was used for immunohistochemistry. Free-floating sections were stained overnight at 4°C with the primary antibodies post-synaptic density protein 95 (1:1000, anti-PSD-95 rabbit polyclonal Ab, Abcam, Cambridge, United Kingdom) or glutamate vesicular transporter 1 (1:500, anti-VGlut1 rabbit polyclonal Ab, Thermo Scientific), and then stained in fluorescent secondary antibody (Cy3-conjugated anti-rabbit secondary antibody 1:200; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, United States) for 2 h at room temperature. For quantification of synaptic puncta, images immunoprocessed for PSD-95 and VGlut1 were acquired with a Leica TCS SL confocal microscope. In each section four images from the PRC were captured and the density of individual puncta exhibiting VGlut1 or PSD-95 immunoreactivity was evaluated as previously described (Guidi et al., 2013 (link)).

The subcellular localization of E-cadherin and ZO-1 proteins was analyzed by confocal fluorescence microscopy using the same antibodies used for western blots. Cells were seeded on 2% gelatin-coated glass coverslips and fixed with 4% paraformaldehyde at room temperature (RT) for 30 min. F-actin staining was performed using rhodamine-conjugated phalloidin as previously described [60 (link)]. To detect ZO-1, in addition to fixation, cells were permeabilized with 0.1% triton X-100 in 0.1% BSA-PBS for 20 min [60 (link)]. Fixed cells were incubated in blocking solution (2 %BSA in PBS, 1 h at RT), followed by an incubation with mouse anti-E-cadherin or rabbit anti-ZO-1 (dilution 1:50) in 0.1% BSA PBS for 1 h at RT. After washing with PBS, cells were incubated for 1 h at RT with FITC-labelled anti-mouse or anti-rabbit Alexa 594 (dilution 1:200) in 0.1% BSA PBS, respectively. After washing with PBS, cells were prepared for visualization by embedding in Vectashield mounting medium with DAPI and visualized in a Leica TCS-SL confocal microscope with a 63X objective.

Cells seeded on 2% gelatin-coated glass coverslips were fixed with 4% paraformaldehyde (PFA) for 20 min. F-actin was stained with rhodamine-conjugated phalloidin (Sigma Aldrich-P1951) as described^{22 (link)}. Samples were visualized in a Leica TCS-SL confocal microscope.

Samples were visualized using a Leica TCS-SL confocal microscope. At least five samples were analysed for each group to assess surface influence on cytoskeletal organization, focal adhesion number, and development and cell morphology. Images were collected and processed using Leica imaging software. At least 50 cells per sample were analyzed. Samples were exposed to the lowest laser power that was able to produce a fluorescent signal for a time interval not higher than 5 min to avoid photobleaching. A pinhole of 1 Airy unit was used. Images were acquired at a resolution of 512 × 512, mean voxel size of 209.20 nm.