C0117

The specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm-thick sections. The sections were stained with periodic acid-Schiff (ab150680, Abcam) for goblet cells or Nissl staining solution (C0117, Beyotime) to identify Nissl bodies according to the manufacturer’s instructions. Subsequently, the slices were dehydrated in a gradient ethanol, cleared in xylene, and covered with neutral resins. The images were acquired by a microscope and analyzed with ImageJ.

After neurobehavioral evaluation, mice (3 animals/group) were anesthetized, and then the brain was removed. The paraffin-embedded mouse hippocampus was cut into 4 mm thick sections. After deparaffinization and rehydration, the sections were stained in Nissl staining solution at 60° C for 30 minutes (C0117, Beyotime Biotechnology, Shanghai, China). The sections are dehydrated with absolute ethanol, xylene is transparent and sealed with neutral gum. The morphology was observed using a multifunctional digital pathology system (APERIO VERSA 8, Leica, Germany).

Neuronal cells in the cortical and hippocampal sections were observed by Nissl staining. The cut brain slices (4 µm) were baked for 1 h in a 60°C oven. The brain slices were dewaxed and placed in a Nissl staining solution (Beyotime, C0117) to react for 15 min. Then, they were washed in distilled water, dehydrated in graded concentrations of ethanol (70%, 80%, 90%, and 100%), cleared in xylene, and finally capped with neutral balsam to seal the sections. Whole-section scanning light microscopy was performed for dark and surviving neurons (digital whole section scanner, Pannoramic MIDI, magnification × 200, × 400). Five random fields (× 200) were selected by blind observers and used to quantify the number of positive cells. Three sections were selected for each animal, and the mean of the cell counts in the right hippocampus and cortex [including cortical, hippocampal cornu ammonis 1(CA1), CA2, CA3, and dentate gyrus (DG) areas] was provided for each animal.

The brain tissues of the mice in each group were carefully removed and rapidly fixed in 4% paraformaldehyde (dissolved in 100 mM PBS) for 24 h. They were then dehydrated in ethanol and embedded in paraffin blocks in sections 3 µm thick using a paraffin tissue slicer (Leica Biosystems, Germany). The brain tissues were subsequently dewaxed with xylene and rehydrated with ethanol, followed by staining with Nith’s solution as per instructions (#C0117, Beyotime, China). The neurons and Nissl bodies in the hippocampus were observed using a biological microscope, and the images were collected and analyzed using ImageJ software.

Nissl staining was performed with a kit according to the manufacturer’s instructions (Beyotime, Cat# C0117, Shanghai, China). Briefly, sections (bregma 0.5 mm to −2.0 mm) were placed in cresyl violet solution at 37°C for 8 min, washed with distilled water twice for 30 s, dehydrated twice with 95 and 100% ethanol for 2 min, cleared three times with xylene for 3 min, and sealed with neutral gum for microscopic examination.

Coronal cryosections were stained with Nissl staining solution (Beyotime, C0117) for 30 min at 37 °C. Then, the cryosections were washed using 95% ethyl alcohol and observed using an optical microscope. A large cell body, with abundant cytoplasm and substantially significant levels of Nissl body, represents a normal neuron. Cells with karyopyknosis or blurred Nissl bodies represented damaged ones.