This article details a method for visualizing amyloid plaques in mouse brain tissue using methoxy X04, a fluorescent dye. The technique allows for precise mapping of plaques within their anatomical context, aiding in the study of Alzheimer's disease pathology.
Begin with a mouse brain tissue section containing the region of interest in a buffer to maintain hydration during imaging.
The tissue is obtained from an Alzheimer's disease mouse model injected with methoxy X04, a fluorescent dye.
Methoxy X04 specifically binds to β-pleated sheet structures in amyloid plaques and retains its fluorescence for imaging and analysis.
Using a fluorescent microscope with a UV filter, excite the methoxy-X04 and capture the emitted fluorescence to visualize fluorescently labeled amyloid plaques against the surrounding tissue.
Without adjusting the microscope stage, capture images of the same region in bright-field mode to detail tissue structure and fluorescence mode to visualize the plaques.
Align the bright-field and fluorescent images using image processing software to precisely map the plaques onto the anatomical structures.
These images provide detailed visualization of amyloid plaques within their structural context to study Alzheimer's disease pathology.
With the aid of a stereotaxic mouse brain atlas, select brain sections containing the region of interest, and place each section into cryoprotectant in a well of a 24-well culture plate. Next, use a disposable pipette to add a droplet of PBS onto a microscope slide and then place the section on the droplet.
Examine the section under a fluorescent microscope to identify regions containing methoxy-X04-labeled A-beta plaques. Without moving the microscope stage, capture images of the regions of interest in bright field as well as in fluorescence mode. Each image must show the same region in both fields to correlate the plaque to the structural region of the tissue section.
Save and name the images taken according to the animal number. Also, record the well number in the plate and the field of the pictures taken. Place the section back into the designated well once the imaging is completed. Examine the next section in the sequence using the same procedure to avoid drying the sections.
To align the images, open the bright field and UV field images for the same ROI in image J. Then, use the mosaic J Plugin to align the edges of the two images. Save the aligned and combined image according to the well number in a folder, with the number of the particular animal. Combine the images from different sections of the same animal to identify and localize the plaques in the region of interest.
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