Measurement of DRG neuron apoptosis by Hoechst 33342 staining

After incubation under different experimental conditions for 24 hours, neuronal apoptosis was evaluated by staining with Hoechst 33342, which directly shows morphological alterations of the nucleus in each neuron. Nuclear DNA was labeled by Hoechst 33342 (Sigma). After termination of the culture procedure at the designed incubation time point, neurons were grown on the surface of coverslips fixed with 4% paraformaldehyde for 10 minutes. Then, the neurons were stained with Hoechst 33342 (5 μg/mL) for 5 minutes in the dark to preserve fluorescence intensity. Apoptotic neurons showed shrunken or condensed nuclei after Hoechst 33342 staining. Neurons with shrunken or condensed nuclei in five visual fields (400×) in the central part of each coverslip were counted as apoptotic neurons in each sample. Simultaneously, all neurons in the same five visual fields were also counted as the total number of neurons. The ratio of apoptotic neurons to total number of neurons was determined.