0.01 m pbs

Morphine was purchased from Shenyang No.1 Pharmaceutical Factory, China. 0.01 M PBS, Triton X-100, naloxone and muscimol were purchased from Sigma, United States. Rabbit anti-c-Fos antibody (Cat#5348) was purchased from Cell Signaling Technology, United States. Goat serum and Alexa Fluor 594-conjugated goat anti-rabbit antibody were purchased from Jackson Immuno Research Laboratory, United States. Biotinylated anti-rabbit secondary antibody was purchased from Vector Laboratories, United States. Other reagents in artificial cerebrospinal fluid (ACSF) were the products of Shanghai Chemical Plant, China.

Anisakis spp. L3 larvae were collected from marine fish as described previously [72 (link)]. Larvae were purified by washing with sterile 0.01 M PBS (pH 7.4; Sigma, St. Louis, MO, USA). The larvae were morphologically identified at the genus level using a stereomicroscope [73 (link)]. Then, species identification of Anisakis spp. nematodes was performed using PCR-restriction fragment length polymorphism (PCR-RFLP) [74 (link)]. Five whole larvae were used to genetic identification of Anisakis species.

The mice were anesthetized by intraperitoneal injection (100 g/ml) of 2% chloral hydrate and 10% urethane-configured anesthetic, and then perfused with 0.01 M PBS (Sigma-Aldrich Inc., St. Louis, United States) for 10 min, followed by perfusion with 4% PFA for 10 min. The brains were removed and post-fixed in 4% PFA solution at 4°C for 24 h. For immunohistochemistry, the brains were cut in 70-μm-thick coronal sections using a vibratome (Leica, VT1200). The brain sections were rinsed in 0.01 M PBS (3 × 10 min), and blocked with 5% (wt/vol) bovine serum albumin in 0.01 M PBS (at 37°C for 2 h). Next, the brain sections were incubated with a primary antibody (at 4°C for 48 h): anti-NeuN (1:800, Rabbit, Abcam, ab7349). After this step, the sections were rinsed with 0.01 M PBS (3 × 10 min), then incubated with a fluorophore-conjugated secondary antibody (1:800, at 37°C for 2 h): Alexa Fluoro-488, Goat anti-Rabbit IgG. Following this procedure, the brain sections were rinsed in 0.01 M PBS (3 × 10 min), and were finally mounted on glass slides, to be imaged with a commercial confocal microscope (Carl Zeiss, LSM710). The RV-labeled neurons and NeuN-positive neurons were manually counted using the Cell Counter ImageJ plug-in (Version 1.48, NIH, United States).

The mean diameter and polydispersity index (PDI) of the NPs were obtained using a Zetasizer (Nano ZS Malvern Instruments, UK) instrument based on the dynamic light scattering (DLS) technique. The measurements were performed on purified NPs by analysing 0.5 mL of the suspension in ultrapure water, placed in a square polystyrene cuvette, at 25 °C. PBS 0.01 M, pH 7.4, Sigma-Aldrich, was used as the diluent in the case of the evaluation of the size after the protein corona formation.