Fluoro jade b

Fluoro-Jade B (FJB; Chemicon, Temecula, CA, USA) is a polyanionic fluorescein derivative that binds with high sensitivity and specificity to degenerating neurons. Briefly, sections were rehydrated in graded ethanol solutions (100 and 70%, 5 min each) and distilled water, incubated in 0.06% KMnO₄ for 30 min, rinsed in distilled water for 2 min, incubated in a 0.001% solution of FJB for 30 min, and observed under a fluorescence microscope (Olympus BX-51; Olympus, Tokyo, Japan) at 450–490 nm.

Cerebellar sections were stained with FluoroJade-B (Chemicon, Temecula, CA), an anionic fluorescein derivative that stains neurons undergoing degeneration. The sections were mounted on glass slides, dehydrated, and stained according to the supplier's manual. The images were acquired using a Zeiss Axiovert 200 imaging microscope. All photographs for comparison were taken under identical image acquisition conditions and uniform adjustments of brightness and contrast.

Fluoro-Jade B (FJB; Chemicon, Temecula, CA, USA) is a polyanionic fluorescein derivative that labels degenerating neurons with high sensitivity and specificity [33 (link)]. Sections were rehydrated in graded ethanol solutions (100% and 70% for 5 min each) and distilled water, incubated in 0.06% KMnO₄ followed by a 0.001% solution of FJB for 30 min each, and observed under a fluorescence microscope (Olympus BX-51; Olympus, Tokyo, Japan) at 450–490 nm.

Mice were perfused with 4% paraformaldehyde dissolved in 0.1 phosphate buffer, pH 7.3 24 hours after the first KA injection, post-fixed overnight in the same fixative, and cryoprotected in 30% sucrose. 30 μm-thick coronal brain sections were obtained in a freezing microtome (Leica, Wetzlar, Germany). Sections containing dorsal hippocampus (Bregma = −1.2 to −1.990 ) were rinsed for 2 h in 0.1 M Tris, pH 7.4, mounted and air dried at room temperature overnight. The next day, sections were pre-treated for 3 min in absolute ethanol, followed by 1 min in 70% ethanol and 1 min in distilled water. They were then oxidized in a solution of 0.06% KMnO4 for 15 min. After three rinses of 1 min each in distilled water, the sections were incubated for 30 min in a solution of 0.001% Fluoro-Jade B (Chemicon) containing 0.01% of DAPI (Sigma) in 0.1% acetic acid. The slides were rinsed in deionized water for 3 min each, dried overnight, cleared in xylene, cover-slipped with Eukitt (Merck, Darmstadt, Germany) and examined using an Olympus (Hamburg, Germany) BX61 epifluorescence microscope. The statistical analysis of the obtained data was performed using Bonferroni post hoc test (Multiple comparison test) using Prism 5.0c (Mac OsX, Grahpad). Data are presented as mean ± standard error of the mean (S.E.M.). A value of ***P < 0.01 was considered statistically significant.

S1P [D-erythro-sphingosine-1-phosphate] was purchased from Avanti Polar Lipid (Alabaster, AL). FTY720 [2-amino-2-[2-(octyl-phenyl) ethyl]-1,3-propanediol hydrochloride] was kindly provided by Novartis AG (Basel). 2,3,5-Triphenyltetrazolium (TTC), 3,3′-diaminobenzidine tetrahydrochloride (DAB), fatty-acid-free BSA (FAF-BSA), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody, anti-β-actin antibody, cresyl violet acetate, and protease inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO). Silicon (Variotime) and Zoletil 50 were obtained from Heraeus Kulzer GmbH (Germany) and Virbac (Carros, France), respectively. Goat polyclonal anti-Iba1 and rabbit polyclonal anti-TNF-α antibodies were purchased from Abcam (Cambridge, UK). Avidin-biotin-peroxidase complex (ABC) kit and Vectashield were purchased from Vector Laboratories, Inc. (Burlingame, CA). Fluoro-Jade B was purchased from Chemicon (Temecula, CA).

Brain sections were washed in 0.01 M phosphate buffered saline (PBS) for a total of 1 h (6 × 10 min), and then mounted on glass slides (Fisher Scientific Superfrost® Plus, Toronto, Canada) using 0.3% gelatin (Fisher Scientific, Toronto, Canada), and were dried overnight. Slides were incubated in 1% sodium hydroxide diluted in 80% ethanol for 5 min. They were then dehydrated in a graded series of ethanol (95% for 3 min, 70% for 3 min, and 50% for 2 min), washed in distilled water (3 × 1 min), and incubated in 0.06% potassium permanganate (Fisher Scientific) for 15 min on a shaker. The slides were washed with PBS (3 × 1 min) and placed in 0.0004% FluoroJade B (Chemicon, Etobicoke, Canada) for 20 min on shaker. Lastly, the slides were washed with PBS (3 × 1 min), dried in an incubator at 37°C for 25 min, cleared in xylene (Caledon Laboratories Ltd., Georgetown, Canada) for 1 min and coverslipped with Depex mounting medium (Electron Microscopy Sciences, Hatfield, USA).

Fluoro-Jade histochemistry was performed to analyze neuronal degeneration in brainstem sections after cochlear ablation (Schmued et al., 1997 (link); Schmued and Hopkins, 2000 (link)). Fluoro-Jade B (Chemicon, Temecula, CA) was used for this purpose, with excitation and emission peaks in distilled water of 480 nm and 525 nm, respectively.
Brain sections from ablated and control animals were mounted onto Superfrost slides and were fully air dried for 20 min. Afterward, the tissue was immersed in 100% ethanol for 3 min, followed by 1 min in 70% ethanol and 1 min in distilled water. Slides were then transferred to a solution of 0.06% potassium permanganate for 15 min, rinsed for 1 min with distilled water, and incubated with 0.001% Fluoro-Jade staining solution for 30 min in the dark. Degeneration was detected in Fluoro-Jade-stained sections under a Leica (Wetzlar, Germany) TCS-SP2 laser scanning confocal microscope.