4.8.1. Hematoxylin and Eosin (H&E) Staining of the Brain, Spinal Cord, and Sciatic Nerve

The H and E staining of the vital organs, including the brain, spinal cord, and sciatic nerve, were performed to investigate the VCR-induced histopathological changes according to previously established protocols [65,66]. The brain, spinal cord, and sciatic nerve samples, intended for histopathological examination, were carefully removed and fixed immediately in 10% formalin. Following fixation, the wet fixed tissues were dehydrated using a graded ethanol series (from 70% to 100%). After dehydration, the tissues were cleaned with a clearing agent (xylene). After clearing, the tissue sections were infiltrated with paraffin wax. After infiltration, the tissues were embedded into a paraffin wax block to enable sectioning on a microtome. The temperature of the embedding paraffin was kept 2–4 °C above the melting point of the wax. The paraffin-embedded tissues were sectioned into 5-µm thickness using a microtome. Following sectioning, the tissue samples were placed on slides. Next, the tissue slides were deparaffinized using absolute xylene (100%), then rehydrated with absolute ethanol, gradient ethanolic concentrations (70–95%), and, finally, with distilled water. After that, the slides were washed in PBS and incubated with hematoxylin for 10 min. After that, the slides were placed in a glass jar under running tap water for 5 min. The slides were then examined under a microscope for nuclear staining, and if the staining was not clear, the hematoxylin timing was increased. The slides were then treated with 1% HCl and 1% ammonia water for a short interval and immediately rinsed with water again. These were then immersed in an eosin solution for 5–10 min, followed by rinsing with water and, finally, air-dried. The slides were then dehydrated using graded ethanol (70%, 95%, and 100%); fixed in xylene; and cover-slipped. The slides were analyzed under a microscope (Olympus, Corporate Parkway, Center Valley, PA, USA), and photos were taken at a magnification of ×4 and ×10 under a microscope.