*4.5. Bielschowsky Silver Staining*

Bielschowsky silver staining is a method that can be used to detect nerve fibers and stain axons, neurofibrils, and senile plaques in the central nervous system. Brain sections were deparaffinized, dehydrated, and then immersed for 15 min in the solution with 20% silver nitrate, preheated at 37 ◦C. Sections were then washed in distilled water, after which they were submerged in silver ammonia solution for 15 min, all at 37 ◦C. Next, slides were developed by placing them in 50 mL of distilled water with eight drops of both ammonium hydroxide and developer stock (8% *v*/*v* formaldehyde, 0.5% *w*/*v* citric acid, 0.1% *v*/*v* nitric acid) for 2 min maximum, i.e., until their color changed to the desired intensity of brown. Following the washing step in distilled water, sections were immersed in 5% sodium thiosulfate for 5 min at room temperature, washed with tap water for 5 min, dehydrated, cleared, and mounted with Entellan*®*.

Microphotographs of the OTs stained with Bielschowsky silver stain were taken at ×400 magnification using an Olympus BX 51 microscope with an Olympus DP 70 digital camera (Olympus, Tokyo, Japan).
