2.3. Immunofluorescence (IFL) and Cell Characterization

Microscopy and IFL were used for SC morphologic characterization. An antibody for the specific SC marker S100 (Dako Agilent, Santa Clara, CA, USA) was used. S100 stains cells of neural origin and is characteristic of SCs in their early stages of development/differentiation. SC cytoskeletons were stained with phalloidin (Sigma-Aldrich). Cells were plated on coverslips, then fixed 20 min in 4% paraformaldehyde (Sigma-Aldrich) and washed in phosphate buffer saline (PBS, Euroclone, Pero, Italy). Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) and blocked for 1 h with 0.25% BSA (Sigma-Aldrich), depending on the host species of the secondary antibody. Primary antibodies to S100 (1:150) and phalloidin (1:300) were applied overnight at 4 °C in a humidified chamber. The following day, slides were rinsed in PBS and incubated in the FITC Alexa-488-conjugated secondary antibody (Thermo Fisher Scientific, Monza, Italy), washed, and mounted using Vectashield^TM plus DAPI for nuclear staining (Vector Laboratories, Oxfordshire, UK). Negative controls lacking primary antibodies were also performed. Confocal laser scanner microscopy was performed by the Zeiss Confocal System and Zen software analysis (Zeiss, Oberkochen, Germany).