Schmued and Hopkins initially described an immunohistochemical method that was able to identify degenerating neurons by the use of an anionic fluorescein derivative stain called Fluoro Jade B [30 (link)]. In the present study, we used a similar yet more sensitive agent, Fluoro Jade C (FJC) to identify TBI-induced degenerating neurons [31 (link)]. Specifically, we used a Fluoro-Jade C from a ready-to-dilute staining kit (Biosensis, TR-100-FJ), with some modification [28 (link)]. Brain sections from the different treatment groups were de-paraffinized, followed by a rehydration step in distilled water for 2 min. The slides were incubated in a 1 in 15 diluted solution of potassium permanganate for 10 min, rinsed in distilled water for 2 min and incubated in a 1:25 diluted FJC solution for 30 min. The slides were then washed and mounted on coverslips with Vecta-shield mounting medium (Vector Laboratories, Burlingame, CA, USA). All sections were observed and photographed with a fluorescence microscope; slides were exposed to an excitation light source of 450–490 nm with the resulting emission at 521 nm recorded and analyzed. The numbers of FJC-positive cells were counted in five randomly selected fields per slide by means of SPOT image analysis software (Diagnostic Instruments, Sterling Heights, MI). The numbers of FJC-positive cells observed on the slides from the different treatment groups were counted and used to generate a mean number per treatment group. The mean numbers of FJC-positive cells were then plotted and used for statistical analysis.
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