Sc301530

Transfected cells were seeded at low density one day prior to fixation. Cells were fixed with 3.7% paraformaldehyde (Sigma) for 10 min, permeabilized with 0.25% Triton X-100 (Sigma) for 5 min, and blocked with 10% BSA (Sigma) for 1 h at room temperature. Samples were incubated overnight at 4 °C with primary antibodies against tubulin (1:200, Abcam ab4074), vimentin (1:200, SantaCruz sc373717), or ezrin (1:200, SantaCruz sc58758). Secondary antibodies anti-Rabbit (1:500, Abcam ab6718), anti-rabbit (1:500, Invitrogen a21206), anti-mouse (1:500, Invitrogen a21202), and anti-mouse (1:500, Invitrogen a31570) were subsequently incubated for 1 h at room temperature. Tetramethylrhodamine (TRITC) or fluorescein isothiocyanate (FITC) coupled phalloidin (1:500, SantaCruz sc301530, sc363791) were used to stain the actin filaments. After staining, coverslips were mounted onto glass slides using ProLong Gold Antifade Mounting medium containing DAPI (Thermo Fisher, Waltham, MA, USA). Images of the fixed samples were obtained using an inverted epifluorescence microscope (Leica DMI4000B, Wetzlar, Germany) with a × 20/0.5 NA objective lens and a CCD camera (Leica DFC300FX, Wetzlar, Germany). Single cells that were not damaged or mitotic were chosen for imaging. Cells were sequentially imaged on TRITC, FITC, and DAPI channels for later analysis.

Primary culture at 15 days (neurons with or without microglial cells) were rinsed with PBS before fixation with 4% paraformaldehyde during 20 min and then washed 3 times in PBS. The poorly adherent microglial cells were lost during these multiple steps of washing. Nerve cells were incubated in blocking buffer (0.05% Triton, 1% Normal Donkey Serum (NDS) and 1% BSA/ovalbumin in PBS/glycine 0.1 M) for 30 min at 4 °C to avoid nonspecific background staining. Neurons were then incubated overnight at 4 °C with mouse monoclonal anti-gliarin antibody diluted in blocking buffer (dilution 1:500, kindly gifted by J. Johansen (Iowa State University, IA, USA)). After 3 washes for 10 min in blocking buffer, the nerve cells were incubated for 1 h at 37 °C with secondary donkey anti-mouse IgG antibody conjugated to Alexa Fluor 488 diluted in blocking buffer (dilution 1:2000, Invitrogen, Carlsbad, CA, USA). Neurons were rinsed with PBS and counterstained with phalloidin tetramethylrhodamine B for 30 min at 4 °C (5 µg/mL, sc 301530, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Finally, after a last PBS washing, cells were mounted on a slide with Dako Fluorescent Mounting Medium (Agilent Dako, Santa Clara, CA, USA). Control experiments were performed following the same immunostaining protocol without the primary antibody incubation.