Visualization of Amyloid-Beta Peptides in Human Cortical Brain Slices

Protocolo

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.  

1. Preparation of Tissue Sections for Imaging Mass Spectrometry (IMS)

1. Processing tissue specimen of a human autopsied brain
   NOTE: Ensure that the sample preparation step for IMS preserves the original state of the tissue. Avoid contamination and post-mortem changes. The following step is crucial.
   1. Obtain human cortical specimens for IMS from brains that were removed, processed, and stored at -80 °C within 8 h postmortem. Take the brain specimen from the occipital cortex of Alzheimer's Disease (AD) patients and age-matched controls.​
2. Preparation of frozen tissue sections
   1. Cut the tissue sections on a cryostat. Place conductive indium-tin-oxide (ITO)-coated microscope glass slides inside the cryostat.
   2. Warm the autopsy brain specimen from -80 °C to -22 °C inside the cryostat.
   3. Attach a new disposable blade to the cryostat for every experiment. Always try to use a clean part of the blade.
   4. Put the frozen autopsy brains on the stage along with a small amount of optimal cutting temperature (OCT) compound (enough to cover the central area of the stage; see Table of Materials).
   5. Choose the conditions for thin sectioning. For IMS, use a thickness of 10–12 μm for human brain sections. For IMS and immunohistochemistry (IHC), cut five to six sections from each tissue sample.
   6. When the blade is just starting to cut the tissue, turn the wheel and "face" the block until all of the tissue is exposed. If there is a small streak or tear across the section, wait a little more in the cryostat until the temperature adjustment automatically fixes it. Count a few seconds before opening the anti-roll with a tissue underneath.
   7. Immediately place the tissue slice on the ITO-coated side of the glass slide. Thaw the tissue slice by putting a finger underneath the slide on the non-ITO-coated side. The tissue will stick to the slide; ensure that the tissue is as flat as possible with no wrinkles. Perform this step at room temperature.
      NOTE: Wear gloves, masks, and a laboratory gown because the human samples may contain biological contaminants. When all sections have been made for the day, clean the cryostat, brushes, and chucks with laboratory wipes and 200 mL of 100% ethanol.
3. Rinsing the tissue sections
   1. Immerse the samples in 40–100 mL of 70% ethanol for 30 s to remove endogenous lipids and inorganic salts. Use a glass staining jar.
   2. Wash the samples with 40–100 mL of 100% ethanol for 30 s, 40–100 mL of Carnoy's solution for 3 min, 40–100 mL of 100% ethanol for 30 s, 40–100 mL of 0.1% trifluoroacetic acid (TFA) for 1 min, and 40–100 mL of 100% ethanol for 30 s. Use a glass staining jar.
      NOTE: Carnoy's solution is a fixative composed of six parts ethanol, three parts acetic acid, and one part chloroform.
   3. Dry in a vacuum for 30 min.
4. Treatment of the tissue sections with a formic acid vapor for a better ionization of the Aβ proteins from autopsy brain tissues
   1. Prepare the oven and incubation glass slide to be used for the subsequent vaporization with 5 mL of 100% formic acid. To achieve a satisfactory acid treatment, keep the air humidity in the incubation glass dish at saturation level throughout this step and keep the temperature at 60 °C. Place the tissue slides in the incubation glass dish while avoiding submersion in the formic acid and treat for 6 min.
   2. Take an optical image of the samples using a film scanner, gel scanner, or a digital microscope, etc. 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.
      NOTE: The most convenient way to take an optical image is to use an office scanner. It is a good idea to save the image in the imaging data storage folder.
   3. 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.
5. Matrix application
   1. Preparation of the matrix solution
      1. In an organic solvent-tolerant microtube, prepare a 10 mg/mL sinapinic acid (SA) solution in 50% acetonitrile (ACN) and 0.1% TFA. Thoroughly dissolve the SA compound by vortexing or brief sonication for 10 min. Store the solution at room temperature until use.
         NOTE: There are three different options for matrix spraying: using an airbrush, an ultrasonic sprayer, or an automatic sprayer.
   2. Spraying the matrix with an airbrush
      1. Perform the operation at a constant room temperature (20–23 °C) and humidity (40%–60%). The parameters to adjust for optimal spraying include the size of the droplet, the amount of mist, the angle and distance between the spray nozzle and the tissue section, and the laboratory temperature and humidity. Adjust these conditions by checking the microscopy results.
         NOTE: As limiting factors include the crystal size and homogeneity of the matrix coverage and the undesirable migration/diffusion of analytes, smaller is better for the drop size. Homogeneity is also the point of inspection.
   3. Spraying the matrix with an ultrasonic sprayer
      1. 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.
      2. Start the preparation by pushing the Start button; usually, prep time is around 90 min. The preparation will be regulated automatically via the monitoring of the matrix layer thickness and wetness. After the preparation is complete, remove the slide and store it in the desiccator for 15 min before reading it in the MALDI instrument.
      3. Clean the sprayer with 2–3 mL of 100% methanol (MeOH) until the spray head appears clean.
         NOTE: A fine mist of matrix droplets is allowed to sink into the tissue by a gravitational sheet. An average droplet size of 20 μm is generated; all droplet diameters are less than 50 μm.
   4. Spraying the matrix with an automatic sprayer
      1. Spray the matrix solution on the tissue surface with an automatic sprayer. A constant flow of heated sheath gas (N₂, set at 10 psi and 75 °C) will be delivered conjointly with the matrix solution spray. Use a solvent pump system (set at 10 psi and 0.15 mL/min) to deliver the matrix solution.
         NOTE: Most importantly, work under a safety cabinet to avoid any inhalation of matrix aerosols. Control the room temperature and humidity to reproduce homogenous matrix crystallization.

2. MALDI-IMS

1. Perform ultra-high-speed mass spectrometry.
   1. Perform high-throughput and high-spatial-resolution imaging experiments with MALDI-IMS equipped with a 10 kHz neodymium-doped yttrium aluminum garnet (Nd:YAG, 355 nm) laser.
   2. For mass spectrometry measurements, define the tissue areas using the MALDI control software and data analysis software.
   3. Acquire spectra in a positive linear mode with a mass range of m/z 2,000–20,000 and a spatial resolution of 20 and 100 µm.
   4. To make the calibration standard, dissolve the peptide calibration standard and the protein calibration standard with a ratio of 1:4 with alpha-Cyano-4-hydroxyl-cinnamic acid (CHCA) in TA30 solution (ACN:0.1% TFA = 30:70) and then dilute it 10x. Place 1 μL of calibration standard on the slide at four different locations.
2. Using molecular histology software (see Table of Materials), overlay multiple single images to find the spatial correlation of various signals, such as different Aβ peptides colocalizing in senile plaques (SPs) and arterial walls.

Materiais

Name	Company	Catalog Number	Comments
Cryostat	Leica Microsystems, Wetzlar, Germany	CM1950
Indium tin oxide (ITO)-coated microscope glass slide	Bruker Daltonics	#237001
Blade (disposable)	Leica Microsystems, Wetzlar, Germany	#117394
O.C.T. Compound	Leica Microsystems, Wetzlar, Germany	FSC 22 Blue
Scanner	EPSON	GT-980
Air-Brush	GSI Creos	PS274
Compressor	MRHOBBY	Mr.Linear compressor L5
Ultrasonic sprayer	Bruker Daltonics	ImagePrep
Automatic sprayer	HTX Technologies	TM Sprayer
Confocal-laser-scanning- microscope	Carl Zeiss Inc.	LSM 700
Ultra high speed MALDI instrument	Bruker Daltonics	rapifleX MALDI Tissuetyper
MALDI control software	Bruker Daltonics	FlexControl 3.8
Molecular histology software	SCiLS, Bremen, Germany	SciLS Lab 2016a
Sinapinic acid (SA)	Nacalai tesque	30494-91
Alpha-Cyano-4-hydroxyl-cinnamic acid (CHCA)	Wako	037-19261
Calibration standard	Bruker Daltonics