Methoxy-X04

This plan only provides a guide, please modify it to meet your specific needs.

In Vitro Imaging Experiment ^[1]

1. Fixation and Section Preparation: Mice were perfused with cold PBS and 4% paraformaldehyde, followed by overnight incubation in 4% paraformaldehyde. Sections were cut with a vibratome to a thickness of 40 μm.

2. Staining Process: Sections were first washed with PBS, then incubated with a solution containing 10 mM methoxy-X04 in 50% DMSO/50% NaCl (0.9%), at a pH of 12, for 10 minutes, followed by two washes with PBS.

3. Fluorescence Detection: Imaging was performed using a laser-scanning confocal microscope. The excitation peak for methoxy-X04 is 750nm, with an emission wavelength range of 460-500 nm.

In Vivo Imaging Experiment ^[2]

1. Solution Preparation: Methoxy-X04 was dissolved in a solution containing 10% DMSO, 45% propylene glycol, and 45% PBS (0.9% NaCl dissolved in 100 mM phosphate buffer, pH 7.4) to prepare a 5 mg/ml methoxy-X04 solution.

2. In Vivo Administration: 24 hours before perfusion, mice were weighed, and methoxy-X04 was administered intraperitoneally at a dose of 10 mg/kg body weight to each mouse.

3. Brain Section Preparation: Mice were euthanized by intraperitoneal injection of 90 mg/kg body weight dose of pentobarbital sodium, brains were extracted, and brain sections were prepared. Each section was placed in a well of a 24-well plate containing sufficient cryoprotectant solution to prevent drying of the sections.

4. Fluorescence Detection: Sections were examined under a fluorescence microscope with a UV filter (excitation wavelength 340 - 380 nm) to identify areas containing methoxy-X04 labeled Aβ plaques.

Precautions:

1. Ensure that all experimental operations comply with laboratory safety standards, especially when handling chemical reagents and conducting animal experiments.

2. Adjust the settings of the fluorescence microscope to obtain the best imaging results.

3. Clean and disinfect the experimental area and equipment before and after experiments to prevent cross-contamination.

4. Detailed records of experimental data, including the weight of the animals and the dosage of drugs, should be maintained.

References:

[1] Tzeng T C , Hasegawa Y , Iguchi R ,et al.Inflammasome-derived cytokine IL18 suppresses amyloid-induced seizures in Alzheimer-prone mice[J].Proceedings of the National Academy of Sciences, 2018, 115(36):201801802.

[2] Bisht K, et al. Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques. J Vis Exp. 2016 Jun 1;(112):54060.