Xylene

To assess the degree of surface hydrophobicity, the microbial adhesion to hydrocarbon (MATH) method was adopted with minor modifications (Rosenberg, Judes & Weiss, 1983 (link); Slizewska, Chlebicz-Wojcik & Nowak, 2021 (link)). LAB strains were washed twice in PBS, harvested by centrifugation (5,000×g, 10 min, 4 °C), and re-suspended in PBS. We measured the optical density of bacterial suspension using spectrophotometry (Biochrom GeneQuant, Cambridge, UK) and adjusted the OD600_nm within the range of 1.0 ± 0.1. Each bacterial suspension (3 mL) was mixed with xylene, ethyl acetate, and n-hexadecane (1 mL, Macklin, Shanghai, China), vortexed for 2 min for emulsion formation, and incubated at 37 °C for 1 h. We then measured the OD600_nm of the aqueous layer. The hydrophobicity was calculated using the following equation:
$$\mathrm{H}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{o}\mathrm{b}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{\%}=[({\mathrm{A}}_0-{\mathrm{A}}_{\mathrm{t}})/{\mathrm{A}}_0]\times 100$$
where A₀ represented the absorbance at time t = 0 h, and A_t represented the absorbance taken at 1 h (Rastogi, Mittal & Singh, 2020 (link)).

The tissue microarray containing 27 samples of NB patients and 5 peripheral nerve tissues was purchased from Biomax (MC642, Derwood, USA). NB xenograft tumor tissues and mouse organ tissues obtained from animal experiments were fixed in 4% paraformaldehyde (Beyotime, Shanghai, China) and embedded in paraffin. Sections were deparaffinized in xylene (MACKLIN, Shanghai, China), rehydrated in graded alcohols (Sinopharm, Beijing, China), and washed in distilled water. Antigens were retrieved by boiling the sections in citric acid-based antigen unmasking solution (Thermo Fisher, Waltham, MA, US) for 30 min, then incubated with primary anti-BRD4 (1:200, ab128874, Abcam, Cambridge, UK) or Ki-67 (1:200, ab15580, Abcam, Cambridge, UK) overnight at 4 °C followed by rabbit specific HRP/DAB detection kit (Cat: ab64261, Abcam, Cambridge, UK) and hematoxylin (Beyotime, Shanghai, China) in compliance with protocols. Staining results were independently evaluated by two experienced pathologists. The total scoring (TS) results were scored by multiplying the percentage of positive cells (P) by the intensity (I). Formula: TS = P × I.

Brain sections (14 μm) were processed through incubation in the following solutions in this order: 100% ethanol (30 s), 95% ethanol (30 s), distilled water (30 s, twice), cresyl violet (Sigma-Aldrich, St Louis, MO, USA) (3–5 min), distilled water (2 min, three times), 50% ethanol (2 min), 95% ethanol (5–30 min), 100% ethanol (5 min, twice), and xylene (MACKLIN, Shanghai, China) (3 min, twice). Thereafter, the sections were mounted with a coverslip.

Shikonin (Meilunbio, S0921AS; purity, >98%) was occupied to treat the experimental group with 20 mg/kg for each mouse, while dexamethasone (Plant Chem Medicine company, Xi’an, China, PCM20356; purity, >98%) was used as positive control in the current study. Xylene (Macklin, X821391; purity, >99%) was utilized to induce a mouse model of acute ear edema. IL-6 (Cell signaling Technology, 12912), IL-1β (Bioteche, AF401-NA), and β-actin (ABclonal, AC026) antibodies were prepared for Western blot assay.

The colon was embedded, frozen at −20°C, and 3 μm sections were cut, water-bath spread, and baked. Sections were stained with hematoxylin (Beyotime, Shanghai, China) for 5 min, followed by rinsing under running tap water for 3 min first and then for 20 s. Sections were then counterstained with eosin (Solarbio, Beijing, China) for 4 min, followed by cleaning with 80% and 95% ethanol (Macklin, Shanghai, China) for 40 s each. Sections were then cleared in xylene (Macklin, Shanghai, China) for 5 s and mounted with neutral balsam. After staining, the sections were observed under a microscope, and the Leica Application Suite was used to collect and analyze the relevant parts of the samples.

Methanol (99.9%, HPLC grade, Concord), xylene (99%, Macklin), TCE (99%, Kermel), palladium acetate (Pd ≥ 47.4%, Mascot), iron nitrate nonahydrate (98.5%, Huaxueshiji Co., Tianjin), trans-dichloroethene (standard grade, AccuStandard) cis-dichloroethene (standard grade, AccuStandard), 1,1-dichloroethene (standard grade, AccuStandard), vinyl chloride (2 g l⁻¹ in methanol, AccuStandard) and CNTs (Chengdu Organic Chemicals Co., Ltd, Chinese Academy of Sciences) were purchased from commercial sources in China and used as received. The chemicals are analytical grade reagents and used without any further purification unless otherwise specified. All the reagents were prepared with deionized water.

For each group, the whole hepatopancreas from 5 shrimps were collected for histological analysis. After being fixed in 4% paraformaldehyde for 24 h, the hepatopancreas tissues were dehydrated using a gradient solution of methanol (25%, 50%, and 75%) with each step lasting 30 min and embedded in paraffin using embedding equipment (Leica Biosystems HistoCore Arcadia, Leica Biosystems, Wetzlar, Germany). The sections were continuously sliced with a fully automated rotary microtome HistoCore AUTOCUT (Leica Biosystems, Wetzlar, Germany) to 4~5 μm thicknesses, spread in a water bath at 40 °C, mounted on glass slides (three to four serial sections per slide), and dried at 37 °C overnight. The wax was removed from the hepatopancreas tissue sections using xylene and ethanol, rehydrated in a decreasing ethanol gradient (100%, 95%, 80%, 70%, 50%, and 30%; 2 min for each step), and stained with hematoxylin–eosin (H&E) (Njjcbio, Nanjing, China). All the stained slides were dehydrated in ethanol and xylene (Macklin, Shanghai, China) for 5 min each, sealed with neutral gum (Solarbio, Beijing, China), and then observed under a BX43 manual light microscope (Olympus, Tokyo, Japan).