Quantifying Amygdalar Neuronal Activity

To evaluate the activity of the neurons in the amygdala, the cfos expression on neurons was calculated in the amygdala. Mice were sacrificed 90 min after fear conditioning. Brian slices (thickness of each slice was 40 μm) separated by 240 μm were immunostained with rabbit anti c-Fos antibody (1:500, 2250S, CST) followed by Alexa Flour 555 conjugated anti-rabbit IgG (1:2000, 4413S, CST) and DAPI (1:5000, CAS: 28718-90-3, Sigma^®). Images were acquired using Leica upright fluorescence microscope (DM6B) using 20× objective and a pixel size of 72 nm. The boundaries of subregions of the amygdala were based on the mouse brain atlas. The c-Fos⁺ neurons were counted by ImageJ software.