Neutral gum

The colons and lungs of mice were collected and fixed in 4% paraformaldehyde (Seven Biotech, Beijing, China). The organs were subjected to ethanol gradient dehydration, liquid paraffin wax wrapping, and wax pourer embedding. Tissue sections were cut using a microtome (Thermo Fisher Scientific, Waltham, MA, USA). Before staining, the tissue samples were dried in an oven (Lichen Technology, Shanghai, China) at 60 °C for 2 h. Tissue slices were dewaxed, stained with hematoxylin and 0.5% eosin (Solarbio, Beijing, China), ethanol gradient-dehydrated, and sealed with neutral gum (Solarbio, Beijing, China).

Paraffin-embedded xenograft sections were used for TDP-43 (Servicebio, China, GB112160, dilution: 1:400), ABHD2 (absin, abs140798, dilution: 1:200), C-caspase3 (Servicebio, GB11532, dilution: 1:500), Bcl-2 (Servicebio, GB113375, dilution: 1:500), and p53 (Servicebio, GB111740, dilution: 1:500) staining analysis. The antibody of Ki67 used in this experiment was purchased from Abcam (ab15580, dilution: 1:200). After deparaffinized and rehydrated, sections were laid in EDTA solution (pH9.0) for antigen retrieval by boiling in microwave oven for 8 min on medium heat. The slides were naturally cooled before treating with 3% H₂O₂ for 10 min. Sections were blocked in 10% normal goat serum for 30 min at room temperature, and then incubated overnight with indicated primary antibody at 4°C. After three washes with PBS, the slides were hybridized with the secondary antibody (Servicebio, GB23303, dilution: 1:200) for 50 min at room temperature. Finally, diaminobenzidine (DAB) (servicebio) was used for color development. After counterstaining with hematoxylin, dehydrate the slides and seal with neutral gum (Solarbio, China). Image J software was used for staining analysis.

To assess myelin loss and the degree of demyelination of the corpus callosum, brain sections were stained with LFB solution. For staining, sections were first dehydrated in 70%-95% gradient ethanol and then incubated with 0.1% LFB solution for 40-50 min at 60°C. Sections were further incubated in 0.5% lithium carbonate solution, followed by differentiation in ethanol solution. Finally, the brain sections were sealed with neutral gum (Cat.: G8590, Solarbio).

Samples were dehydrated in gradient alcohol, fixed in neutral‐buffered formalin and embedded in paraffin. They were prepared into 4‐6‐μm‐thick tissue sections and then dewaxed. For H&E staining, hematoxylin and eosin were stained, rinsed by water, then dehydrated, and sealed by neutral gum as the manufacturer's instructions (Solarbio, Beijing, China). For IHC staining, antigen retrieval was performed by pressure cooking at 100°C in 0.01M Sodium Citrate buffer for 20 minutes. Slides were then treated with 3% H₂O₂ in methanol. After blocking, the samples were incubated with the first PLK1 antibody diluted 1:500 at 37°C for 1 hours. The rest procedure of immunohistochemistry was performed as the manufacturer's instructions (Absin, Shanghai, China), and the final results were determined by two pathologists independently.

The volatile oil of CX was obtained from Guangzhou Aroma Master Chenxiang Technology Co., Ltd. (Guangzhou, China) and the volatile oil of MX was obtained from Changshengyuan Medicinal Materials Development Co., Ltd. (Mianyang, China). Fluoxetine hydrochloride (FH) was purchased from Lilly Suzhou Pharmaceutical Co., Ltd. (Suzhou, China). The commercial enzyme-linked immunosorbent assay (ELISA) kits for adrenocorticotropic hormone (ACTH), corticosterone (CORT), 5-hydroxytryptamine (5-HT), dopamine (DA), norepinephrine (NE) and acetylcholine (Ach) were purchased from Jiangsu Enzymatic Immunity Industry Co., Ltd. (Yancheng, China). β-Actin and 5-HT_1A antibodies were purchased from Abcam (Cambridge, UK). The reagents (i.e., ethanol, chloroform, isopropanol, H₂O₂ and xylene) were of analytical reagent grade and were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Biyuntian Biotechnology Co., Ltd. (Shanghai, China). The SYBR Green PCR kit was purchased from Thermo Fisher Scientific (China) Co., Ltd. Neutral gum and phosphate buffered saline (PBS) solution were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).