Immunocytochemistry Protocol for Cell Culture Analysis

Immunocytochemistry was performed as previously described^{23 (link)}. Briefly, cell cultures were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. After blocking overnight with 4% albumin, the cells were incubated overnight with anti-GFAP (1:400; Dako [Z0334], Carpienteria, CA), anti-GLT-1 (1:1000; Alpha diagnostic [GLT11-A], San Antonio, TX), anti-vimentin (1:1000; Sigma Aldrich [V6630], St. Louis, MO) or anti-NeuN (1:50; EDM Millipore [MAB377], Billerica, MA) at 4°C, followed by PBS washes and incubation with a specific secondary antibody (Jackson ImmunoResearch, West Grove, PA) conjugated with Alexa Fluor® 488 (green staining, [111-545-003]) or 594 (red staining, [315-585-003]) for 1 h at room temperature. For all the immunostaining-negative controls, reactions were performed omitting the primary antibody. No reactivity was observed when the primary antibody was excluded. Cell nuclei were stained with 0.2 mg/mL of 4′,6′-diamidino-2-phenylindole (DAPI; EDM Millipore [268298], Billerica, MA). The cells were visualized with a Nikon inverted microscope and the images were transferred to a computer with a digital camera (Sound Vision Inc.).