Mouse brain sections were immunostained with mouse NeuN antibody. Immunolabeling was visualized by using Alexa Fluor 555 dye. Sections were rinsed three times with PBS and one time with water before performing the Fluoro-Jade B procedure. Briefly, sections stained with NeuN were mounted on Superfrost Plus microscope slides and air dried overnight. The sections were rinsed in distilled water for two minutes to rehydrate and transferred to a solution of 0.06% potassium permanganate for ten minutes. The sections were then rinsed in distilled water for two minutes and placed in a 0.0004% Fluoro-Jade B solution made by adding 4 ml of a 0.01% stock solution of Fluoro-Jade B to 96 ml of 0.1% acetic acid. After 20 minutes in the Fluoro-Jade B staining solution, the stained slides were thoroughly washed in distilled water, dehydrated, and cover slipped.
