Quantifying Neuronal Degeneration with Fluoro-Jade B

Fluoro‐Jade B immunofluorescent data were used from a previously published study (Qin & Crews, 2012b (link)). Briefly, paraffin‐embedded human OFC sections were deparaffinized, washed in PBS, and immunostained with mouse anti‐NeuN (1:100; Millipore). Immunolabeling was visualized using Alexa Fluor 555 dye. Sections were washed in PBS and water, then transferred to a solution of 0.06% potassium permanganate for 10 min. Sections were then rinsed in distilled water for 2 min and placed in a 0.0004% Fluoro‐Jade B solution made by adding 4.0 ml of a 0.01% stock solution of Fluoro‐Jade B (Millipore) to 96 ml of 0.1% acetic acid. After 20 min in the Fluoro‐Jade B solution, stained slides were thoroughly washed in distilled water, dehydrated, and cover slipped (Qin & Crews, 2012b (link)). Fluorescent intensity of Fluoro‐Jade B was quantified using BioQuant Nova Advanced Image Analysis software.