Fluoro-Jade

DNA damage was evaluated by the terminal deoxynucleotidyltransferase (TdT)-mediated UTP nick end labeling (TUNEL) assay. Degenerating cells were stained by the Fluoro-Jade dye. 

Fluoro-Jade staining was performed as described in Schmued and Hopkins (2000) with modifications (Butler et al. 2002). The sections were wet mounted onto microscope glass slides and air-dried for 30 min at 37°C in an oven. Then, the sections were pretreated for 5 min in absolute alcohol, followed by 3 min in 70% ethanol, 3 min in 50% ethanol, and 5 min in distilled water. The slides were then immersed in a solution of 0.05% KMn04 for 30 min at room temperature, and stained for 30 min in a solution of 0.001% Fluoro-Jade B (Chemicon, Temecula, CA, USA) in 0.1% acetic acid. Finally, the slides were then rinsed in distilled water, dried, cleared in xylene, and coverslipped. 

Cells positive for NeuN, TUNEL, and Fluoro-Jade were evaluated in grids of 800 pm x 500 pm (CAl) or 800 pm x 1,000 pm (neocortex and striatum). Areas were determined by public domain software (ImageJ http //rsb.info.nih.gov/ij). The number of positive cells was divided by the respective area (total area or frame area). Thus, single-labeled cells for any marker were quantified densitometrically (cells/mm2). Densities were averaged to obtain a mean density value for each region/animal group. 

Fig. 3A-D Postischemic damage in CA1 on day 4. A Staining for the TUNEL assay showing numerous positive cells. B Statistical analysis of TUNEL+ cells per frame of 0.4 mm2 in CA1 p < 0.001, one-way ANOVA followed by Tukey-Kramer post hoc. C Staining for Fluoro-Jade demonstrating similar pattern to the TUNEL stain. D Statistical analysis of Fluoro-Jade+ cells per frame of 0.4 mm2 in CA1 p < 0.001, one-way ANOVA followed by Tukey-Kramer post hoc. Scale bar = 200 pm...

Fig. 3C, D). The density of Fluoro-Jade+ cells on day 4 (80 4 per frame Fig. 5F) was compatible with that of TUNEL+ cells on day 4 (92 4 per frame Fig. 3B, D). In contrast to CA, DG remained negative for Fluoro-Jade at all time-points (data not shown). 

Carlson, K., Ehrich, M., 2004. Organophosphorus compound-induced delayed neurotoxicity in white leghorn hens assessed by Fluoro-Jade. Int. J. Toxicol. 23, 259-266. 

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