Quantifying Neurodegeneration via Fluoro-Jade B

To assess for neurodegeneration one day after KA-SE, FluoroJade B (FJB) (Histochem, #1FJB, Jefferson, AR) was used to label and quantify degenerating neurons (Schmued et al., 1997 (link)) according to manufacturer’s instructions with modifications (Rojas et al., 2014 (link)). Briefly, hippocampal sections were mounted and dried on SuperFrost Plus microscope slides. Once dried, sections were sequentially immersed with 80% EtOH, 70% EtOH, and ddH₂O for 2 minutes each. Sections were incubated with 0.06% KMnO₄ (dissolved in ddH₂O) for 10 minutes and rinsed twice with ddH₂O for 2 minutes each to remove all traces of KMnO₄. Sections were incubated in a 0.0004% solution of Fluoro-Jade B for 30 minutes covered from light. Sections were then rinsed thrice with ddH₂O for 1 minute each and air dried for 24 hours while covered from light. Sections were cleared with xylenes for 5 minutes and coverslipped with DPX (Electron Microscopy Sciences, Hatfield, PA) mounting medium. FJB labeled neurons were visualized and imaged using a Leica DM6000B epifluorescent upright microscope with filters for fluorescein or FITC at 10X and 20X magnifications. Representative images were collected from dorsal hippocampal CA fields. A standardized region of interest (ROI) was drawn around CA3 cell bodies in the stratum pyramidale (SP) from 20X magnified images. Fluoro-Jade B positive cells were quantified using DotDotGoose (Ersts, American Museum of Natural History, Center of Biodiversity and Conservation, version 1.5.3). Cell counts are represented as the total number of Fluoro-Jade B positive cells in each representative section for each animal.