visualization of amyloid-beta peptides in human cortical brain slices

Visualization of Amyloid-Beta Peptides in Human Cortical Brain Slices

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.

visualization-of-amyloid-beta-peptides-in-human-cortical-brain-slices

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

EoE Sub Articles

eoe sub articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. &#160;
1. Preparation of Tissue Sections for Imaging Mass Spectrometry (IMS)
<ol>
	<li>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.
	<ol>
		<li>Obtain human cortical specimens for IMS from brains that were removed, processed, and stored at -80 &#176;C within 8 h postmortem. Take the brain specimen from the occipital cortex of Alzheimer&#39;s Disease (AD) patients and age-matched controls.&#8203;</li>
	</ol>
	</li>
	<li>Preparation of frozen tissue sections
	<ol>
		<li>Cut the tissue sections on a cryostat. Place conductive indium-tin-oxide (ITO)-coated microscope glass slides inside the cryostat.</li>
		<li>Warm the autopsy brain specimen from -80 &#176;C to -22 &#176;C inside the cryostat.</li>
		<li>Attach a new disposable blade to the cryostat for every experiment. Always try to use a clean part of the blade.</li>
		<li>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&#160;Table of Materials).</li>
		<li>Choose the conditions for thin sectioning. For IMS, use a thickness of 10&#8211;12 &#956;m for human brain sections. For IMS and immunohistochemistry (IHC), cut five to six sections from each tissue sample.</li>
		<li>When the blade is just starting to cut the tissue, turn the wheel and &#34;face&#34; 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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Rinsing the tissue sections
	<ol>
		<li>Immerse the samples in 40&#8211;100 mL of 70% ethanol for 30 s to remove endogenous lipids and inorganic salts. Use a glass staining jar.</li>
		<li>Wash the samples with 40&#8211;100 mL of 100% ethanol for 30 s, 40&#8211;100 mL of Carnoy&#39;s solution for 3 min, 40&#8211;100 mL of 100% ethanol for 30 s, 40&#8211;100 mL of 0.1% trifluoroacetic acid (TFA) for 1 min, and 40&#8211;100 mL of 100% ethanol for 30 s. Use a glass staining jar. 
		NOTE: Carnoy&#39;s solution is a fixative composed of six parts ethanol, three parts acetic acid, and one part chloroform.</li>
		<li>Dry in a vacuum for 30 min.</li>
	</ol>
	</li>
	<li>Treatment of the tissue sections with a formic acid vapor for a better ionization of the A&#946; proteins from autopsy brain tissues
	<ol>
		<li>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 &#176;C. Place the tissue slides in the incubation glass dish while avoiding submersion in the formic acid and treat for 6 min.</li>
		<li>Take an optical image of the samples using a film scanner, gel scanner, or a digital microscope,&#160;etc.&#160;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:&#160;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Matrix application
	<ol>
		<li>Preparation of the matrix solution
		<ol>
			<li>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:&#160;There are three different options for matrix spraying: using an airbrush, an ultrasonic sprayer, or an automatic sprayer.</li>
		</ol>
		</li>
		<li>Spraying the matrix with an airbrush
		<ol>
			<li>Perform the operation at a constant room temperature (20&#8211;23 &#176;C) and humidity (40%&#8211;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.</li>
		</ol>
		</li>
		<li>Spraying the matrix with an ultrasonic sprayer
		<ol>
			<li>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.</li>
			<li>Start the preparation by pushing the&#160;Start&#160;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.</li>
			<li>Clean the sprayer with 2&#8211;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 &#956;m is generated; all droplet diameters are less than 50 &#956;m.</li>
		</ol>
		</li>
		<li>Spraying the matrix with an automatic sprayer
		<ol>
			<li>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 &#176;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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. MALDI-IMS
<ol>
	<li>Perform ultra-high-speed mass spectrometry.
	<ol>
		<li>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.</li>
		<li>For mass spectrometry measurements, define the tissue areas using the MALDI control software and data analysis software.</li>
		<li>Acquire spectra in a positive linear mode with a mass range of m/z 2,000&#8211;20,000 and a spatial resolution of 20 and 100 &#181;m.</li>
		<li>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 &#956;L of calibration standard on the slide at four different locations.</li>
	</ol>
	</li>
	<li>Using molecular histology software (see&#160;Table of Materials), overlay multiple single images to find the spatial correlation of various signals, such as different A&#946; peptides colocalizing in senile plaques (SPs) and arterial walls.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Indium tin oxide (ITO)-coated microscope glass slide</td>
			<td>Bruker Daltonics</td>
			<td>#237001</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade (disposable)</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>#117394</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>FSC 22 Blue</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanner</td>
			<td>EPSON</td>
			<td>GT-980</td>
			<td></td>
		</tr>
		<tr>
			<td>Air-Brush</td>
			<td>GSI Creos</td>
			<td>PS274</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressor</td>
			<td>MRHOBBY</td>
			<td>Mr.Linear compressor L5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic sprayer</td>
			<td>Bruker Daltonics</td>
			<td>ImagePrep</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic sprayer</td>
			<td>HTX Technologies</td>
			<td>TM Sprayer</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal-laser-scanning- microscope</td>
			<td>Carl Zeiss Inc.</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra high speed MALDI instrument</td>
			<td>Bruker Daltonics</td>
			<td>rapifleX MALDI Tissuetyper</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI control software</td>
			<td>Bruker Daltonics</td>
			<td>FlexControl 3.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular histology software</td>
			<td>SCiLS, Bremen, Germany</td>
			<td>SciLS Lab 2016a</td>
			<td></td>
		</tr>
		<tr>
			<td>Sinapinic acid (SA)</td>
			<td>Nacalai tesque</td>
			<td>30494-91</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha-Cyano-4-hydroxyl-cinnamic acid (CHCA)</td>
			<td>Wako</td>
			<td>037-19261</td>
			<td></td>
		</tr>
		<tr>
			<td>Calibration standard</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Ikegawa, M. et al. <a href="https://app.jove.com/v/57645">Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry.</a> J. Vis. Exp. (2019)
The video demonstrates a procedure for preparing human cortical brain slices from Alzheimer&#39;s disease patients for analyzing the deposition of amyloid-beta (A&#946;) peptides using Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry (MALDI-IMS). The steps include preparing the slides, treating the tissue to enhance ionization, and analyzing the spatial distribution of amyloid-beta peptides.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. &#160;
1. Preparation of Tissue ...

Take a glass slide containing a cortical slice of brain tissue collected from an Alzheimer&#39;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&#39;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.

15 次观看. 人皮质脑切片中淀粉样蛋白-β 肽的可视化

视频: 人皮质脑切片中淀粉样蛋白-β 肽的可视化 - 实验

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

科研

JoVE Journal

生物化学

使用偏振敏感双光子显微镜表征球晶

Polarization-Sensitive Two-Photon Microscopy for a Label-Free Amyloid Structural Characterization

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

作者聚焦:使用偏振敏感双光子显微镜对复杂生物结构进行无创成像 

This paper describes how polarization-sensitive two-photon microscopy could be applied to characterize the local organization within label-free amyloid superstructures-spherulites. It also describes how to prepare and measure the sample, assemble the required setup, and analyze the data to obtain information about the local organization of amyloid fibrils.

偏振敏感双光子显微镜用于无标记淀粉样蛋白结构表征

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue ...

化学

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor &#947;-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-&#916;E (N&#916;E; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress &#947;-secretase cleavage of APP-C99 in CG cells, but not N&#916;E in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for &#947;-secretase modulators, and these responses are in stark contrast to the pan-inhibition of &#947;-secretase induced by DAPT. Our studies provide direct evidence that &#947;-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

We have successfully generated two substrate-specific &#947;-secretase assays. Both cell-based assays presented here are designed to quantify &#947;-secretase enzymatic activities via the output of firefly luciferase reporters.

基于细胞荧光检测平台的γ-分泌介导的淀粉样前体蛋白和缺口裂解的定量测定

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

Visualization of Amyloid-Beta Peptides in Human Cortical Brain Slices

成績單