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1. Fixation and Staining of Cryopreserved Brain Tissue Sections
<ol>
	<li>To preserve RNA integrity, clean all instruments to be used with RNase cleaning solution. Proceed with the fixation and staining protocol inside a fume hood.</li>
	<li>Prepare the described fixative solutions in clean RNase-free 50 mL tubes. Make all solutions with RNase-free water on the day of the staining.</li>
	<li>Same day the laser microdissection will be performed, prepare 100%, 95%, 70%, and 50% ethanol solutions. Keep the solutions in tightly closed tubes at room temperature.</li>
	<li>Prepare 4% Cresyl violet and 0.5% eosin Y in 75% ethanol solution. Vortex the solution vigorously for 1 min and filter them through a 0.45 &#181;m nylon filter to eliminate traces of undissolved powder.</li>
	<li>Place the tissue slides into a container with 95% ethanol for 30 s. Transfer slides to the tube containing 75% ethanol; leave slides there for 30 s.</li>
	<li>Transfer slides to 50% ethanol and leave them there for 25 s. At this point, the OCT will be dissolved. Transfer the slide to 4% Cresyl violet solution for 20 s and then transfer to 0.5% eosin Y solution for 5 s.</li>
	<li>Take the slide out of the dye solution and blot the slide dry with a filter paper. Then, place the slides in 50% ethanol for 25 s. Transfer the slides to 75% ethanol for 25 s. Transfer the slides to 95% ethanol for 30 s. Transfer the slides to 100% ethanol for 60 s.</li>
	<li>Rinse the slide with xylene. Transfer them to a container with xylene and wait 3 min.</li>
	<li>Prepare mounting medium (e.g., Pinpoint gum) in RNase-free water. To mount mouse brain sections, dilute the mounting medium in RNase-free water at a ratio of 1:10.</li>
	<li>Dry the slides on an RNase-free surface at room temperature for 10 s. Before the xylene dries, proceed to mounting the slides with the tissue sections.</li>
	<li>Gently disperse mounting solution on top of the tissue on the slide with a sterile and RNase-free thin paintbrush. Wait 10&#8211;20 s and then immediately transfer the tissue slides to the microscope microdissection platform. 
	NOTE: The ratio of mounting medium to RNase-free water used for mounting varies depending on the tissue of interest. The mounting medium/water ratio preserves glioma tissue morphology for laser microdissection without affecting the RNA integrity.</li>
</ol>

<table><tbody><tr><td>Cresyl Violet Acetate</td><td>Sigma Aldrich&amp;nbsp;</td><td>C5042</td><td></td></tr><tr><td>Eosin Y&amp;nbsp;&amp;nbsp;</td><td>Sigma Aldrich</td><td>E4009</td><td></td></tr><tr><td>RNaseZap RNase Decontamination Solution</td><td>Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>PEN Membrane Glass Slide (2 &amp;micro;m)</td><td>Lieca&amp;nbsp;&amp;nbsp;</td><td>1150518</td><td></td></tr><tr><td>Pinpoint Solution</td><td>Zymo Research</td><td>D3001-1</td><td></td></tr><tr><td>Tissue-Plus O.C.T. Compound&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>23-730-571</td><td></td></tr></tbody></table>

cryopreserved tissue section fixation and staining: a procedure to process frozen brain tissue sections for laser microdissection

Source: Comba, A. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/60939/laser-capture-microdissection-glioma-subregions-for-spatial-molecular">Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion.</a>&#160;J. Vis. Exp.&#160;(2020)
This video presents the fixation and staining method to process frozen mouse brain tumor tissue. The processed tissue helps differentiate and obtain a specific population of cells during subsequent laser capture microdissection.

Cryopreserved Tissue Section Fixation and Staining: A Procedure to Process Frozen Brain Tissue Sections for Laser Microdissection

Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, ...

To differentiate specific cell populations in a heterogeneous mouse brain tumor tissue for their subsequent isolation, begin by taking a glass slide carrying the cryopreserved brain tumor tissue section of interest.
Sequentially incubate the tissue in decreasing concentrations of ethanol. Ethanol dissolves the embedding medium that has penetrated the tissue and fixes the tissue while maintaining nucleic acid integrity.&#160;
Additionally, ethanol rehydrates the tissue, enabling even staining during the subsequent steps.
Now, treat the tissue with Cresyl violet. Cresyl violet - a basic dye - stains acidic cellular components such as nucleic acids.
Thereafter, counterstain the tissue with Eosin Y. Eosin Y - an acidic dye - stains basic cellular components such as cytoplasmic proteins.
This differential staining step helps distinguish between the cytoplasm and other cellular structures, enabling the identification of cells of interest.
Post staining, immerse the tissue in increasing concentrations of ethanol to dehydrate the tissue and avoid excessive tissue distortion.
Next, wash the tissue with xylene. Xylene displaces and removes ethanol from the tissue section.
Finally, add a suitable mounting solution onto the tissue section to preserve tissue morphology.
The processed tissue section is ready to be used for laser microdissection - a process that allows for precise isolation of specific cells from the tissue.

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Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

In vitro studies

1.3K Views. Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion. J. Vis. Exp. (2020) This video presents the fixation and staining method to process frozen mouse brain tumor tissue. The processed tissue helps differentiate and obtain a specific population of cells during subsequent laser capture microdissection. 

Video: Cryopreserved Tissue Section Fixation and Staining: A Procedure to Process Frozen Brain Tissue Sections for Laser Microdissection - Concept

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

JoVE Journal

Neuroscience

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Medicine

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

Cryopreserved Tissue Section Fixation and Staining: A Procedure to Process Frozen Brain Tissue Sections for Laser Microdissection

Transcript

Cryopreserved Tissue Section Fixation and Staining: A Procedure to Process Frozen Brain Tissue Sections for Laser Microdissection

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Free-floating Immunostaining of Mouse Brains