The immunohistochemistry method was as previous described (59). Briefly, brains were immersion-fixed in Bouin's fixative overnight at 4°C, processed in paraffin, and 4 μm brain sections were prepared on a microtome. Deparaffinized sections were incubated with anti-FoxO1 or anti-FoxO3a, labeled with horseradish peroxidase-conjugated anti-rabbit IgG, and counter-stained with Hoechst 33,258. Immuno-fluorescence in brain sections was viewed with a digital confocal microscope and photographed using a 100× objective. Negative controls of FoxO1 and FoxO3a immunostains were obtained from Brain FoxO1 knockout mouse and FoxO3a-deficient mouse, respectively.
