Detection of active caspase-3 by immunofluorescence. Primary cortical cells were cultured for 7 days in 96-well microplates and then treated with or without 1 mM glutamate for 3 h and 6 h. The cells were fixed and then incubated first with primary rabbit anti-cleaved caspase-3 antibody and then with FITC-goat anti-rabbit secondary antibody as described under "Methods". After aspiration, the cells were washed twice with PBS + saponin and the relative fluorescence measured using a Labsystem Fluroskan Accent FL microplate reader (excitation 490 nm; 520 nm emission). The bars depict relative fluorescence units from 3 measurements (± SEM). P < 0.05 compared to control untreated cells.