Xylene based synthetic mount

The Luxol fast blue Staining was performed according to manufacture protocol (Eurolab) to stain myelin sheath. Briefly, the slides were deparaffinized through 2 times incubation in xylene for 10 min each. They were transferred to a series of alcohol for rehydration in the order of 100% ethanol 2 times, 90% ethanol, 70% ethanol of 5 min each. The slides were hydrated by incubation in distilled water for 5 min and then incubated in Luxol fast blue solution overnight at 37°C to stain the myelin sheath. The stain was differentiated by dipping in lithium carbonate solution, followed by dipping in 70% ethanol and finally rinsed in distilled water. They were incubated in Cresyl Echt Violet for 2–5 min and then rinsed in distilled water. The slides were then transferred to a series of alcohol for dehydration in the order of 70% ethanol, 90% ethanol and 100% ethanol 2 times for 2 min each. Finally, the slides were incubated for 5 min in xylene and tissues were mounted with xylene-based synthetic mount (Thermo Scientific) with cover slides. Luxol Fast Blue stained images were captured with Olympus DP74 microscope digital camera attached to Olympus BX43 microscope.

The slides were incubated 2 times in xylene (Eurolab, Brussels, Belgium) for 10 mins each. They were then transferred to a series of ethanol (Honeywell, Charlotte, North Carolina, USA) for rehydration in the order of 100% ethanol 2 times, 90% ethanol, 70% ethanol of 5 min each. The slides were incubated in distilled water for 5 min and then incubated in Shandon Harris Hematoxylin (Thermo Scientific) for 3–7 min to stain the nuclei. The slides were then transferred to a staining jar with running tap water until the slides were clear. They were incubated in Shandon Eosin (Thermo Scientific) for 2–4 min and transferred to a series of alcohol for dehydration in the order of 70% ethanol, 90% ethanol and 100% ethanol 2 times of 2 min each. Finally, the slides were incubated for 5 min in xylene and tissues were mounted with xylene-based synthetic mount (Thermo Scientific) with cover slides. Hematoxylin and Eosin (H & E) stained images were captured with Olympus DP74 microscope digital camera attached to Olympus BX43 microscope.