Name: Video: Isolamento delle fibrille amiloidi dall'estratto di tessuto cerebrale - Concept
Uploaded: 2025-01-30T14:42:53.000+00:00
Duration: 1 min 26 s
Description: 61 Views. Fonte: Upadhyay, A. et al., Purificazione biochimica e caratterizzazione proteomica dei nuclei di fibrille amiloidi dal cervello. J. Vis. Exp. (2022) Questo video mostra il protocollo per isolare le fibrille amiloidi dall'estratto di tessuto cerebrale di topo utilizzando la centrifugazione in gradiente di densità e la digestione di proteine non amiloidi.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and amyloid purification
NOTE: Ideally, amyloid fibrils should be isolated from freshly dissected brain regions. However, this method also works well with snap- or flash-frozen brain tissues. Below is a brief outline of snap-freezing brain tissues for storage for use at a later time.
<ol>
	<li>Brain tissue harvesting and storage: Dissect mouse brains and quickly harvest the amyloid-laden regions (i.e., cortex and hippocampus), followed by snap freezing in liquid nitrogen or a dry ice alcohol bath. 
	NOTE: Snap freezing helps to preserve the structural attributes of the tissue&#39;s constituents. The mice are euthanized by isoflurane and cervical dislocation. It is advised to avoid using CO2 since that can compromise brain proteome integrity.</li>
	<li>Post-dissection, cut, and store fresh tissues in small blocks (e.g., 5 mm x 5 mm), as this facilitates more efficient homogenization before amyloid extraction. Transfer the tissue to the sterile cryogenic vial, tighten its cap, and rapidly submerge.</li>
	<li>Keep tissues frozen in ultracold conditions (i.e., -80 &#176;C or liquid nitrogen). If extraction is to be performed a few days later, store the tissues at -20 &#176;C and avoid freeze and thaw cycles. Thaw the frozen tissues on wet ice when ready for extraction and purification. 
	&#8203;NOTE: Direct contact of the tissues with liquid nitrogen can cause tissue and protein damage. Note that an alcohol bath can remove permanent markers from the tube labels.</li>
</ol>
2. Enrichment of sodium dodecyl sulphate (SDS) insoluble material
NOTE: Perform all the steps on ice and centrifuge at 4 &#176;C, unless stated otherwise. Manufacturers and catalog numbers of chemicals and instruments are provided in the Table of Materials.
<ol>
	<li>Start with freshly dissected or snap-frozen brain tissue regions (thawed on ice) placed into a 2 mL tube containing 6-8 ceramic beads and freshly prepared ice-cold homogenization buffer (1 mL for 0.25-1 g of wet tissue mass).</li>
	<li>Transfer the tubes to the bead mill homogenizer and grind the tissue contents at 4000 rpm, with two cycles of 30 s each and an interval of 30 s in between. 
	NOTE: Alternatively, motorized stirrer or homogenizer systems can be used to grind and homogenize the tissues.</li>
	<li>Add 9 mL of ice-cold homogenization buffer to the 1 mL of brain whole tissue homogenate in 15 mL tubes, seal with laboratory wax film strips, and rotate end-to-end overnight at 4 &#176;C to ensure robust solubilization. 
	NOTE: To remove unwanted large cellular debris and lipids, on the next morning, spin the tubes at 800 x g at 4 &#176;C for 10 min and collect the supernatant in a fresh tube. Resuspend the remaining pellet in 2 mL of ice-cold homogenization buffer, mix well, spin again for 10 min at 4 &#176;C, and combine the two supernatants.</li>
	<li>Slowly add solid sucrose to the tissue extract suspension to a final concentration of 1.2 M, mix well, and centrifuge at 250,000 x g for 45 min at 4 &#176;C.</li>
	<li>Carefully remove and discard the supernatant using a pipette. Resuspend the pellet in 2 mL of homogenization buffer containing 1.9 M sucrose by triturating, followed by centrifugation at 125,000 x g for 45 min at 4 &#176;C. 
	NOTE: The buffer volume can be adjusted based on the amount of material recovered from the previous step. In general, 10 volumes of homogenization buffer (V/V) is ideal for this step.</li>
	<li>Collect the top white solid layer using a pipette, transfer it to a fresh tube, and solubilize in 1 mL of ice-cold wash buffer by pipetting up and down several times, without introducing air bubbles.</li>
	<li>Apart from the top layer, the pellet is also enriched with amyloid fibrils. For a higher yield, combine the two fractions and proceed. Carefully remove the middle aqueous layer using a pipette and discard.</li>
	<li>Centrifuge the combined fractions at 8000 x g for 20 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Add 1 mL of ice-cold digestion buffer to the washed pellet, resuspend, and incubate at room temperature (RT) for 3 h.</li>
	<li>Centrifuge at 8000 x g for 20 min at 4 &#176;C and remove the supernatant using a pipette.</li>
	<li>Resuspend and wash (same as Step 2.8.) the digested pellet two times in 1 mL of ice-cold Tris buffer.</li>
	<li>Resuspend the washed pellet in 1 mL of solubilization buffer containing 1% SDS and 1.3 M sucrose by pipetting up and down. Centrifuge quickly at 200,000 x g for 60 min at 4 &#176;C. 
	NOTE: Although the amyloid fibrils are highly resistant to detergents and denaturants, very long exposures to the detergent (1% SDS) may affect the integrity of the fibrils or remove the tightly bound proteins, which will compromise subsequent analyses. Therefore, immediately after resuspending the pellets, proceed to centrifugation.</li>
	<li>Save the pellet and increase the volume of the remaining supernatant by adding 50 mM Tris buffer (in ratio 1:0.3) to reduce the sucrose concentration from 1.3 to 1 M and centrifuge one more time at 200,000 x g for 45 min at 4 &#176;C.</li>
	<li>Dissolve the two pellets in 100 &#181;L of Tris buffer containing 0.5% SDS and pool for amyloid purification. Visible pellets are small and should appear opaque and off-white (see Figure 1A).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protease Inhibito</td>
			<td>Thermo Scientific</td>
			<td>164535</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma-Aldrich</td>
			<td>C0130</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0521</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>K54 Tissue Homogenizing System Motor</td>
			<td>Cole Parmer</td>
			<td>Glas-Col 099C</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>BR-8101P-E</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Precellys 24 tissue homogenizer</td>
			<td>Bertin Instruments</td>
			<td>P000062-PEVO0-A</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>74255</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21R Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002446</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>15568025</td>
			<td>Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
	</tbody>
</table>

isolation of amyloid fibrils from brain tissue extract

Source: Upadhyay, A. et al., <a href="https://app.jove.com/v/63816">Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.</a> J. Vis. Exp. (2022)
This video demonstrates the protocol for isolating&#160; amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

<img alt="Figure 1" src="/files/ftp_upload/22972/22972_Figure1.jpg" /> 
Figure 1: Confirmation of amyloid extraction using biochemical staining and imaging of amyloid fibrils. (A) Enriched amyloid-containing SDS insoluble pellet appears opaque off-white in color. (B) Congo red staining of SDS soluble supernatant and SDS insoluble pellet containing purified amyloid blotted on 0.45 &#181;m nitrocellulose membrane; BCA readings were use...

Isolation of Amyloid Fibrils from Brain Tissue Extract

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and ...

Take a mouse brain-tissue extract containing non-amyloid proteins and amyloid fibrils, an insoluble protein aggregate.
Add solid sucrose and mix, increasing the solution&#39;s density.
Transfer it and centrifuge to separate denser proteins from debris.
Discard the supernatant, resuspend the pellet in a higher sucrose concentration buffer, mix and centrifuge.
Collect the top layer containing a few amyloid fibrils in a fresh tube, add a wash buffer, and mix.
Now, discard the middle layer and transfer the amyloid fibrils-enriched pellet to the tube containing the top layer fraction for maximum isolation.
Centrifuge the combined fractions and discard the supernatant. Add a digestion buffer and incubate to degrade non-amyloid proteins exclusively.
Centrifuge and remove the supernatant. Wash the pellet to remove residual digested proteins.
Resuspend the pellet in a sucrose-based solubilization buffer containing detergent that solubilizes the remaining non-amyloid proteins.
Centrifuge, remove the supernatant, and resuspend the isolated amyloid fibrils.&#160;

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61 Views. Fonte: Upadhyay, A. et al., Purificazione biochimica e caratterizzazione proteomica dei nuclei di fibrille amiloidi dal cervello. J. Vis. Exp. (2022) Questo video mostra il protocollo per isolare le fibrille amiloidi dall'estratto di tessuto cerebrale di topo utilizzando la centrifugazione in gradiente di densità e la digestione di proteine non amiloidi. 

Video: Isolamento delle fibrille amiloidi dall'estratto di tessuto cerebrale - Concept

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Un Tailored HPLC purificazione protocollo che i rendimenti di elevata purezza beta-amiloide 42 e beta amiloide 40 peptidi, in grado di Oligomer Formazione

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

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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.

Imaging dell'amiloide tessuti macchiati con luminescenti oligotiofeni coniugati di Hyperspectral confocale microscopia e fluorescenza 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 ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

Isolation of Amyloid Fibrils from Brain Tissue Extract

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Isolation of Amyloid Fibrils from Brain Tissue Extract