Visualization of Amyloid-Beta Peptides in Human Cortical Brain Slices

Take a glass slide containing a cortical slice of brain tissue collected from an Alzheimer's patient. The tissue contains insoluble amyloid-beta deposits within the parenchyma and cerebral capillaries.

Immerse the slide in diluted alcohol to remove lipids and salts.

Wash with absolute alcohol to dehydrate the tissue, then apply a fixative to preserve the tissue architecture.

Vacuum dry the slide, removing any moisture.

Treat with formic acid vapor in a humidified environment to break down amyloid-beta aggregates into smaller soluble peptides.

Using an optical scanner, generate images of the tissue's structural features to interpret data from the imaging mass spectrometry.

Spray a matrix solution over the specimen.

Using a mass spectrometer, apply a laser that the matrix absorbs to ionize the peptides. A detector then identifies them based on their mass-to-charge ratio.

Analyze the data to produce images, revealing the distribution of amyloid-beta peptides in the tissue parenchyma and cerebral capillaries.

To rinse the tissue sections, immerse the samples in 40 to 100 milliliters of 70% ethanol in a glass staining jar for 30 seconds to remove endogenous lipids and inorganic salts. Wash the samples using the washing sequence listed in the text protocol. Then, dry the samples in a vacuum for 30 minutes.

Now, treat the tissue sections with a formic acid vapor for a better ionization of the amyloid-beta proteins from autopsy brain tissue. To do so, prepare the oven at 60 degrees Celsius and an incubation glass dish with 5 milliliters of 100% formic acid. Keep the air humidity in the incubation glass dish at saturation level.

Place the tissue slides in the incubation glass dish while avoiding submersion in the formic acid and treat for six minutes. Take an optical image of the samples using a film scanner, gel scanner, or a digital microscope. Perform this step at room temperature.

The alignment of the optical image of the samples is necessary when the sample target is placed inside the instrument. Usually, it will not be possible to recognize the tissue section underneath the matrix layer. To correlate the optical images with the samples, make guide marks that are visible both in the optical image and underneath the matrix layer in the camera optic. The easiest way is to spot at least three correction fluid marks around the sample before taking the optical image.

To spray the matrix with an ultrasonic sprayer, remove the tissue to be sprayed from the desiccator, and place it in the chamber. Make sure the tissue is not covering the sensor window. Start the preparation by pushing the Start button. Usually, prep time is around 90 minutes.

The preparation will be regulated automatically via the monitoring of the matrix layer thickness and wetness. Alternatively, to spray the matrix solution on the tissue surface with an automatic sprayer, use a solvent pump system set at 10 PSI and 0.15 milliliters per minute to deliver the matrix solution.

A constant flow of heated sheath gas will be delivered conjointly with the matrix solution spray. Perform high throughput and high spatial resolution imaging experiments with MALDI-IMS. For mass spectrometry measurements, define the tissue areas using the MALDI control software and data analysis software.

Acquire spectra in a positive linear mode with a mass-to-charge ratio range of 2,000 to 20,000 and a spatial resolution of 20 and 100 microns. To make the calibration standard, dissolve the peptide calibration standard in the protein calibration standard in a 1-to-4 ratio with CHCA in TA30 solution, and then, dilute it 10 times.

Place 1 microliter of calibration standard on the slide at four different locations. Using molecular histology software, overlay multiple signal images to find the spatial correlation of various signals, such as different amyloid-beta peptides co-localizing in senile plaques in arterial walls.