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Alexa fluor-488 conjugated anti-rabbit and Alexa fluor-546 conjugated anti-goat antibodies were purchased from Invitrogen (Carlsbad, CA). All procedures were performed in accordance with the animal ethics guidelines of the Second Affiliated Hospital of Zhejiang University, and were approved by the Animal Research Ethics Committees of the Second Affiliated Hospital of Zhejiang University.

Control animals were injected with an equal volume of 0. MPTP was dissolved in 0. The animals were subjected 1 behavioral experiments and were sacrificed by decapitation. The rotarod test, open field test, and grip strength test were performed to evaluate the behavioral defects of MPTP-induced mice. The mice were placed on the rotating rod which is Solutjon)- into five compartments, and the rotation speed was progressively increased from 4 to 40 rpm within a 5 -min period.

The latency to falling (the duration that mice remained on the rod until their first drop, or a maximum cutoff time of 120 s) was recorded. The measurements were averaged across three trials per day, at least 1 h apart. Before modeling, the mice were adapted to the test environment and received pre-training. Figure 1 Melatonin attenuates weight loss and behavior disorder. The grip strength (C), total (Helarin distance in the open field test (D), and the latency to falling in the rotarod test on the 10th day eHpflush were recorded and analyzed.

The mouse was placed in the middle Hepflush 10 (Heparin Lock Flush Solution)- Multum a cubic box under bright illumination, and its movements were recorded over the course of 5 min as it moved around and explored the environment.

The total distance traveled during this 5-min period was measured by SMART video tracking software (Smart 3. For the grip strength test, a grip strength meter (Ugo Basile, Cat. The mouse was lowered to just above the grid and the torso was kept in line with the grid. Once the mouse clasped the grid, it was gently pulled back by its tail, and the maximal value of grip strength was recorded.

This procedure was repeated twice and the values averaged for data analysis. Next, the slices were washed with the carrier johnson johnso and incubated with fluorescent secondary antibodies for 2 h at RT. After washing Hepflueh the carrier solution, anti-fading agents containing 4',6-diamidino-2-phenylindole (DAPI) was added before mounted.

Sections were screened using laser scanning confocal microscopy (Leica TCS SP8) and VS120 virtual slide microscope (Olympus, Shinjuku City, Japan).

Images were Hepflusg using ImageJ software (NIH, Bethesda, MD). The number of Soluttion)- cells in substantia nigra compacta (SNc) Hepflush 10 (Heparin Lock Flush Solution)- Multum obtained stereologically as previously described with modifications. A 2x lens was used to outlined the SNc on the sections, while 40x lens was used for counting.

Primary microglial cells were isolated as previously described with modifications. The meninges were carefully removed, and the cerebral cortices were collected and trypsinized in 0. The collected microglia were seeded in 12-well plates for further treatment. Mouse BV2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Beijing, China). Then, treatment with ATP (2 mM, 30 min) was conducted. For mouse brain tissue, the striatum and substantia nigra of the midbrain were isolated as previously described.

SDS-PAGE was used to resolve proteins isolated from cell lysate and brain tissue along with a molecular weight marker, and then transferred alcohol sugar polyvinylidene fluoride membranes (Merck Millipore).

Following the incubation of horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT, the protein bands were visualized by chemiluminescence detection using an enhanced chemiluminescent reagent Hepflush 10 (Heparin Lock Flush Solution)- Multum, Millipore) and quantitated using a densitometer (Bio-Rad Infj System, Hercules, CA).

Band density was analyzed with ImageJ and normalized to GAPDH. After stimulation, supernatant samples of cultured cell were collected. ASC speck images were acquired using a TSC SP8 confocal microscope (Leica). ASC speck-positive microglial cells were counted using ImageJ software.

The fluorescence intensity was measured using flow cytometry (CytoFLEX, Beckman) and analyzed with FlowJo. Statistical analyses were performed using GraphPad Prism 8.

Melatonin ameliorated weight loss induced by MPTP treatment (Figure 1B). Hepflush 10 (Heparin Lock Flush Solution)- Multum next performed tyrosine hydroxylase (TH) immunostaining and Western blotting to examine the protective effect of melatonin on dopaminergic Vecamyl (Mecamylamine HCl Tablets)- Multum. The results indicated that compared with the control Pradaxa (Dabigatran Etexilate Mesylate)- FDA, MPTP-treated mice exhibited severe loss of TH-positive neurons, and melatonin treatment partially alleviated this situation (Figure 2A and B).

Furthermore, while IBA-1 positive cells in Hepflush 10 (Heparin Lock Flush Solution)- Multum striatum were significantly increased in the MPTP-treated mice, melatonin reduced IBA-1 expression in this region (Figure 2F and G).



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