Immunohistochemistry and immunofluorescence
Mice were transcardially perfused with saline followed by infusion of 4% paraformaldehyde through the left ventricle. Following this, the brain was dissected from the skull, and tissue was placed in 4% PFA and left overnight at 4oC, after which the brain tissues were preserved in 30% sucrose solution until the tissue sank. The fixed tissue was sliced using a sliding microtome and brain sections were rinsed and incubated overnight with primary antibodies. For immunohistochemical staining, sections were incubated with anti-tyrosine hydroxylase (1:400; Abcam, USA, cat: ab75875), followed by enzyme-conjugate IgG (1:50; Beyotime, China) secondary antibody.
For immunofluorescence staining, BV2 was incubated with primary antibody against TLR4 (1:100; Santa Cruz, USA, cat: sc-293072). After several washings, cells were incubated with goat anti-mouse secondary antibody CY3 (1:8000; Abcam, UK) in a dark room. The images were taken using a confocal fluorescence microscope (Leica, Germany). IHC stained cells were examined using a brightfield microscope (Leica Germany).
Liquid Chromatography tandem mass spectrometry (LC-MS/MS)
Appropriate amounts of tissue were exhaustively chopped and homogenized. Following this, tissues were transferred into a centrifuge tube, and 3 mL of 10% sodium carbonate solution and 10 mL ethyl acetate were added to the tube. Tissues were subsequently homogenized by shaking for 10 min at 4oC. After centrifugation (6000 r/min for 10 min), the upper layer organic phase was transferred into a pear-shaped bottle and then subjected to rotary evaporation to dry at 40oC. The residue was dissolved in 1 mL 50% acetonitrile solution and this solution was cooled for 30 min and centrifuged at 16,000 r/min for 5 min. An appropriate amount of the supernatant was filtered through a 0.22μm membrane and then analyzed by LC-MS/MS. The devices used in this study are a Waters Acuity UPLC liquid chromatography (Waters, USA), an AB SCIEX 5500 Qtrap-MS mass spectrography (AB SCIEX, USA), and an Acquity UPLC HSS T3 chromatographic column (1.8 μm x 2.1 mm x 100 mm; Waters, USA).