Twelve weeks after being plated onto glass coverslips, neural cell lines from CTLs and ADs were fixed using 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized using 0.2% triton X-100 (Sigma-Aldrich) in PBS for 10 min. Following a 1-hr block using 5% donkey serum (Jackson ImmunoResearch, West Grove, PA), cultures were incubated for 24–48 hours at 4° with the following primary antibodies diluted in 5% donkey serum in PBS: mouse anti-GFAP (1:500, Millipore, Brillerica, CA), rabbit anti-MAP2 (1:500, Millipore), or rabbit anti-TBR1 (a forebrain glutamatergic neuron marker; 1:1000, ProteinTech Group, Chicago, IL, incubation included 0.1% triton X-100). Cells were then incubated at room temperature for 2-hours in donkey anti-mouse alexa fluor 594 (1:1000, Thermo Fisher Scientific) and donkey anti-rabbit alexa fluor 488 (1:1000, Thermo Fisher Scientific) secondary antibodies diluted in 3% donkey serum in PBS for visualization. TBR1+ cells as a percentage of the total cell population were quantified from 6 control and 6 alcoholic neural cell lines (derived from 5 control and 6 alcoholic donor subjects).
