Name: Vídeo: Coloração com tioflavina-S da agregação beta-amilóide em tecido cerebral de camundongo hipoperfundido - Experimento
Uploaded: 2025-03-04T15:34:57.000+00:00
Duration: 1 min 21 s
Description: 60 visualizações. Coloração com tioflavina-S da agregação beta-amilóide em tecido cerebral de camundongo hipoperfundido

thioflavin-s staining of amyloid-beta aggregation in cerebral hypoperfused mouse brain tissue

Thioflavin-S Staining of Amyloid-Beta Aggregation in Cerebral Hypoperfused Mouse Brain Tissue

Take a brain tissue section from a mouse subjected to mild chronic cerebral hypoperfusion, or MCCH, and injected with amyloid-beta peptides.
MCCH is characterized by reduced blood flow to the brain. This limits oxygen and nutrient supply to the endothelial cell layer of cerebral blood vessels, damaging the cells.
The damage increases the vascular wall permeability, allowing the injected amyloid-beta peptides to accumulate in the wall and form plaques.
Wash the section with a buffer to remove residual chemicals.
Incubate with thioflavin S, a fluorescent dye that binds to the amyloid plaques.
Wash the stained section with decreasing alcohol concentrations to remove any unbound dye and rehydrate the tissue.
Place the section in the buffer to maintain hydration.
Mount the section using a mounting medium that preserves the dye&#39;s fluorescence intensity.
Under a fluorescence microscope, examine the presence of fluorescently labeled amyloid deposits to confirm MCCH-induced amyloid-beta aggregation.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Cerebrovascular Diseases

60 visualizações. Coloração com tioflavina-S da agregação beta-amilóide em tecido cerebral de camundongo hipoperfundido

Vídeo: Coloração com tioflavina-S da agregação beta-amilóide em tecido cerebral de camundongo hipoperfundido - Experimento

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

Administração exógena de agregados de microssomas associados alfa-synuclein para os neurônios primários como um modelo de célula poderosos de formação de fibrilas

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

JoVE Journal

Neurociência

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Um modelo in vitro para estudar a agregação Tau usando transdução mediada por Lentivirus de neurônios humanos

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Um método de limpeza de tecido hidrofóbico para tecido cerebral de rato

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Thioflavin-S Staining of Amyloid-Beta Aggregation in Cerebral Hypoperfused Mouse Brain Tissue

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