*4.4. Fluoro-Jade C Staining*

The slides were deparaffinized in xylene, rehydrated in ethanol and water, and then treated for 10 min with a 0.06% potassium permanganate solution. Sections were rinsed twice with distilled water (dH2O) for 1 min and incubated in 0.0001% Fluoro-Jade C (Chemicon, Millipore, Billerica, MA, USA) staining solution for 20 min in the dark. After that, they were washed in dH2O thrice per minute and dried on a hot plate on 50 ◦C for 20 min. Sections were dehydrated in xylene two times for 10 min, mounted in *Entellan®* (Merck Millipore, Billerica, MA, USA), and coverslipped. Stained sections were examined by epifluorescence microscopy using the appropriate light filter cube (Olympus BX 51 microscope with Olympus DP 70 digital camera, Olympus, Tokyo, Japan).

Quantification of Fluoro-Jade C intensity in the OT was done on microphotographs taken at ×400 final magnification; for each animal, two images were used for the analyses. Within each microphotograph, three ROIs of 0.0057 mm<sup>2</sup> were analyzed. By subtracting the background fluorescent intensity from those ROIs, we could determine only degenerating axons within that field.
