Immunocytochemistry of Neuronal and Glial Cells

Cells cultured on coated coverslips were washed with PBS and fixed with 4% PFA at room temperature for 15 min. Then cultures were washed three times in PBS with 0.05% Triton X-100, followed by blocking at room temperature for 30 min in TBS-Blotto (0.15 M NaCl, 20 mM Tris-HCl, pH 7.5, 4.5% non-fat dry milk) with 0.1% Triton X-100. Cells were incubated with anti Tuj1 (1:1000, T8578, Sigma-Aldrich) and GFAP antibody (1:1000, #PA1-10019, ThermoFisher Scientific) for 1 h at room temperature with shaking, washed three times, and incubated with corresponding Alexa Fluor secondary antibodies (A11029, A11012, ThermoFisher Scientific) covered with foil for 1 h. Cells were rinsed twice, stained with 300 nM DAPI for 5 min, and rinsed twice. Coverslips were mounted with mowiol and imaged using the Lionheart FX automated microscope. Coverslips were coded, and the experimentalist was blind to their assignments. In total, 15–25 fields from 3–5 coverslips were scanned under 20× objective and counted using ImageJ [29 (link)].