*4.8. Cresyl-Violet*

Cresyl-violet (Nissl) staining was used to detect the effects of repetitive mTBI on the number of neurons in the investigated nuclei of the visual system. Deparaffinized and rehydrated slides were stained with 0.1% cresyl-violet acetate (Sigma Aldrich, St Louis, MO, USA) by incubation for 10 min at room temperature. Differentiation of the brain tissue sections was done by immersing the slides in 95% alcohol with glacial acetic acid. Finally, brain sections were dehydrated in alcohol, cleared in xylene, and mounted with Entellan*®*.

Microphotographs of the cresyl-violet stained sections were taken at approximately −2.46 from bregma for the LGN and at −3.52 from bregma for the SC [85], at ×400 magnification, using an Olympus BX 51 microscope equipped with an Olympus DP 70 digital camera (Olympus, Tokyo, Japan). With the help of the ImageJ software, neuronal density estimation in the investigated nuclei was carried out by a blind investigator using the random simple counting method. Two to four random ROIs were taken from at least two serial cuts of the selected areas and used to estimate the number of the neurons. Only cells with visible nuclei were counted.
