Tcsnt sp2 confocal microscope

At the time of sacrifice, 8 cm segment of the distal ileum was flashed with PBS, filled with 4% PFA and submerged in the same fixative for 1 h at 22°C. Tissues were then washed in PBS (3 × 10 min) and stored at 4°C (Brun et al., 2013 (link)). Whole mounts were prepared from 1 cm long specimen under a dissecting microscope (Zeiss, Germany) by peeling off the longitudinal muscle layer containing the myenteric plexus (LMMP). LMMP were gently stretched, pinned down on a wax support, washed twice with PBS containing 0.5% Triton-X100, incubated in blocking buffer (2% bovine serum albumin, 0.5% Triton-X100 in PBS) and then stained at 4°C for 16 h with primary antibody (Table 1). Fluorescent labeled secondary antibodies (Table 1) were used to detect immune-complexes using Leica TCSNT/SP2 confocal microscope.

The h-osteoblast cells were cultured on functionalized glass coverslips for 24 h at 37 °C. Cells were then washed twice in phosphate buffer saline solution (PBS) and fixed in 4% w/vol buffered PFA for 10 min at room temperature. After extensive washing, cells were permeabilized by incubation with 0.5% vol/vol Triton X-100 in PBS for 15 min at room temperature. Unspecific binding sites were blocked by incubation with 1% w/vol bovine serum albumin (Merck) and then cells were labeled with anti-phospho FAK rabbit monoclonal antibody (clone 31H5L17, Thermo Fischer Scientific) for 2 h. Cells were washed and incubated with the goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488 (Thermo Fischer Scientific) for 45 min at room temperature. The samples were washed three times in PBS and mounted with Prolong Antifade kit (Life Technologies). Samples were imaged using Leica TCSNT/SP2 confocal microscope.

Cultured neurons were washed and fixed in PFA (4% w/vol) for 20 min at room temperature. Cells were then permeabilized with 0.5% Triton X-100 in PBS for 15 min and incubated at room temperature for 1 h with rabbit polyclonal anti-peripherin antibody (Millipore, Milan, Italy). Following extensive washes in PBS, cells were incubated for 1 h at room temperature with TRITC labeled secondary antibodies (Molecular Probe, Milan, Italy). Nuclei were stained with TOTO-3 iodide (Molecular Probe). Samples were then analysed using Leica TCSNT/SP2 confocal microscope. All microscope parameters were set to collect images below saturation and were kept constant during acquisition.
Number of peripherin positive cells was recorded within a minimum of 3 randomly selected fields covering 1.254 mm² from five independent experiments. The percentage of peripherin positive cells was calculated on the number of total nuclei stained with TOTO-3 iodide. Axonal length was measured in stained neuronal cells by using ImageJ 1.40 g (National Institutes of Health; Bethesda, MD, USA). For each experimental condition, 10 cells in 8 images obtained from at least three separate staining sessions were considered. Quantitative analysis was performed manually by using a counting grid, as previously reported^{43 (link)}.