*4.6. Luxol Fast Blue Staining*

LFB staining of the OT was performed to determine the degree of myelination. Following deparaffinization and rehydration, brain sections were stained with LFB solution at 56 ◦C overnight. The next day, slides were rinsed with 95% ethanol and distilled water, after which they were differentiated in the lithium carbonate solution for 10–15 s, and then immersed briefly in the 70% ethanol three times. Following that, slides were washed in distilled water, dehydrated through rising ethanol concentrations, cleared in xylene, and mounted with Entellan*®*.

LFB-stained sections were photographed at 200× magnification using an Olympus BX 51 microscope with an Olympus DP 70 digital camera (Olympus, Japan).

Myelin densities on LFB stained photographs were quantified by using the ImageJ software (NIH, Bethesda, Md, USA), according to the protocol described by Underhill et al. [86] with modifications suggested by Khodanovich et al. [87]. Briefly, mean intensities of the red channel in a region of interest (ROI), i.e., in the OTs, were measured from RGB images as a quantity characterizing the complementary blue channel saturation. The background mean intensity of the red channel was also measured on each photograph in ROIs outside the brain tissue, which served for the calculation of the background correction factor. LFB optical density (in %) was calculated for each ROI according to the following formula: LFB density = 100 × (1 − (red channel intensity/background intensity)).
