Cresyl violet solution

Eight male Wistar rats were used to determine the distribution of GAL-ir nerve fibers in the dura mater. They were anesthetized with isoflurane (Fujifilm Wako Pure Chemical Corporation, Japan) until respiration was markedly suppressed and transvascularly perfused with 50 mL saline followed by 500 mL Zamboni fixative^{42 (link)}. The brain with the meninges was dissected and immersed in the same fixative. Then, the samples were immersed overnight in a phosphate-buffered saline containing 20% sucrose and frozen with dry ice. The whole brain was frontally and serially sectioned at 8 µm and thaw-mounted on silane-coated glass slides (n = 4). The caudal 1/3 portion of the brain, including the brainstem and cerebellum, was also horizontally sectioned in four animals. The 480-µm interval sections of the brain were stained with 0.2% cresyl violet solution (Sigma-Aldrich, USA) and subjected to immunohistochemistry to demonstrate the structure of the dura mater and the distribution of GAL and other substances within it.

The brains of the experimental animals were transcardially perfused with fixative solution containing 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.3 and postfixed in the same fixative overnight at 4°C. Then, they were immersed in a cryoprotectant containing 30% sucrose (Merck, Germany) solution at 4°C for 72 h. Serial sections of the frozen tissues were cut at 20 μm thick using cryostat (Thermo Scientific™ HM525 Cryostat). All sections were placed on slides coated with 0.3% gelatin containing 0.05% aluminium potassium sulfate (Sigma-Aldrich, USA). Following this step, the sections were stained with 0.2% cresyl violet solution (Sigma-Aldrich, USA) for 8 minutes, rinsed with distilled water, and dehydrated with 70%, 95%, and 100% alcohols, respectively (RCI LabScan, Thailand). Then, the sections were immersed in xylene 2 times for 5 minutes each and mounted with DPX mountant (Merck, Germany). The determination of neuron density was performed based on the stereotaxic coordinates from the rat brain atlas as the following: anteroposterior 2.5-4.5 mm and mediolateral 0.2-1.0 mm [21 ] under the Olympus light microscope model BH-2 (Japan) at 40x magnification. Results were expressed as density of neurons per 255 μm².