Fluoro jade c

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 mm² were analyzed. By subtracting the background fluorescent intensity from those ROIs, we could determine only degenerating axons within that field.

Brain sections (20-μm cryostat brain sections) were stained with Fluoro-Jade C to detect neuronal death as described previously.^{18 (link)} Briefly, brain slides were incubated in 100% ethanol (3 min), 70% ethanol (1 min), distilled water (1 min), and then a solution containing 0.0004%, Fluoro-Jade C (Merck Millipore, Darmstadt, Germany), and 0.1% acetic acid for 30 min. Sections were washed three times in distilled water, then dried overnight before being immersed in xylene (3 × 2 min) and mounted with DPX neutral mounting medium (Sigma-Aldrich, St. Louis, MO). Three coronal brain sections (200 μm between slices, −1.2 and −2.2 mm from bregma) from each mouse were imaged with a fluorescence microscope (BX63; Olympus, Tokyo, Japan). Fluoro-Jade C–positive cells were counted using the CellSens Count and Measure Solution module (Olympus) in the perilesional region of the ipsilateral hemisphere by an investigator blind to the treatment.