Rabbit polyclonal anti peripherin antibody

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)}.

C2C12 myotubes were incubated with 10 ​μg/ml Alexa594-conjugated α-bungarotoxin (Invitrogen, 1938422). The cells were then fixed in 4 ​% paraformaldehyde (PFA) for 15 ​min. Before and after fixation, the cells were washed with phosphate buffered saline (PBS) three times. For peripherin staining in addition to α-bungarotoxin staining, the cells were treated with PBS containing 2 ​% goat serum (Fujifilm Wako Pure Chemical, WDR2410) and 0.1 ​% Triton X-100 for 1 ​h. After washing with PBS, the cells were incubated with rabbit polyclonal anti-peripherin antibody (1:300, Millipore, 2972430) overnight at 4 ​°C, and with Alexa488-conjugated goat anti-rabbit IgG (1:100, Invitrogen, SH251139) for 1 ​h at room temperature. The staining was observed with an IX71 fluorescence microscope (Olympus) and analyzed by CellSens software (Olympus). The total area and the total intensity of AChR clusters were blindly evaluated using MetaMorph software (Molecular Devices).