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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Section Screening for the Presence of Methoxy-X04-stained Plaques
- With the aid of a stereotaxic mouse brain atlas, select brain sections containing the region of interest, for instance, the hippocampus CA1 as used in the present example. Place each section into a well within a 24-well culture plate containing enough cryoprotectant solution so as to prevent the sections from drying out.
- Examine each section successively to avoid drying out the sections using the following procedure:
- Add a droplet of phosphate-buffered saline (PBS) to a microscope slide with a disposable pipette and place the section on the droplet.
- Examine the section under a fluorescent microscope to identify regions containing methoxy-X04 labeled Aβ plaques.
Note: methoxy-X04 can be easily visualized using a fluorescence ultraviolet (UV) filter (excitation 340 - 380 nm).
- Capture images of regions of interest (ROI) in bright field as well as in fluorescence mode without moving the microscope stage, since each image must show the same region in both fields in order to correlate the presence of the plaques directly 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. For example, Ex: 9978A1B; Animal number: 9978; Well number: A1; Field: Bright. Place the section back in its designated well once the imaging is completed.
Note: In the end, for each section examined, two pictures are obtained, one in bright field and another in UV field. - Open the two images of the same ROI (one in bright field and the other in UV field) in Image J, and using the MosaicJ plugin, align the edges of the two images and save the aligned and combined image according to the well number it came from.
- Save the picture in a separate folder with the number of the particular animal. Combine the images to identify and localize the plaques in the tissue section and have a full view of the whole section in the two different fields (bright and UV).
- After the screening process is completed, store the examined sections at -20 °C in a 24-well culture plate containing cryoprotectant until immunostaining or further processing is carried out.