Name: Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue
Uploaded: 2023-04-30T14:00:24
Description: Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, ...

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Sectioning Frozen Brain Tumor Tissues
<ol>
	<li>Label 2 &#181;m polyethylene naphthalate (PEN) slides with the sample information. Tissue sections will be placed directly on these slides following sectioning.</li>
	<li>Set the temperature of the cryostat chamber between -20 to -24 &#176;C. Before sectioning, place the sample block in the cryostat chamber and let it equilibrate to the temperature in the chamber for 30&#8211;60 min.</li>
	<li>Clean the cryostat chamber and the knife holder with 100% ethanol and spray the brushes to be used with RNase cleaning solution. Working inside the cryostat chamber, remove the mold and attach the OCT block containing the brain to the cryostat specimen disk with OCT. Place the block in the disk holder and align the block with the knife blade.</li>
	<li>Install a disposable blade into the sectioning holder.</li>
	<li>Section the brain at 10 &#181;m thickness. Make sure there are no streaks or scratch lines in the tissue. Using a paintbrush, cautiously flatten and uncurl the tissue onto the cutting surface.</li>
	<li>Carefully mount the tissue containing the brain sections onto RNase-free PEN glass slides. Flip the positively charged glass slides with the fingers in direction of the tissue and smoothly press the glass slide down towards the tissue section. 
	NOTE: Temperature of hands will help the tissue attach to the glass.</li>
	<li>After mounting the brain sections onto the slides, keep the slides in a box inside the cryostat chamber and then store them at -80 &#176;C. Never keep the slides at room temperature. 
	NOTE: Folding of the tissue and tearing are common. For accurate posterior analysis, it is important to minimize these artifacts.</li>
</ol>

frozen mouse brain tissue sectioning: a procedure to obtain thin frozen tissue sections from frozen murine brain tissue

Source: Comba, A. et al. <a href="https://www.jove.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> J. Vis. Exp. (2020)
In this video, we demonstrate the sectioning of a frozen mouse brain tumor tissue using a cryostat. The frozen brain tissue sections so obtained are stored at low temperatures until further analysis.

Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue

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

To obtain cryosections or thin frozen tissue sections of a mouse brain, first, take a frozen block of mouse brain embedded in a suitable embedding medium.&#160;
Transfer the frozen block to a cryostat chamber and allow the block&#8217;s temperature to equilibrate with the set temperature of the cryostat for subsequent sectioning procedure.
Then, use embedding medium to mount the frozen tissue block to the base of the cryostat specimen disc.
Place the specimen disc into the specimen head of the cryostat.&#160;
Additionally, secure a blade in the blade holder of the cryostat.
Using the external handwheel, adjust the position of the specimen head to align the tissue block with the blade.
Once the tissue block and blade are correctly oriented, move the handwheel to initiate sectioning, trimming off the unwanted region of the tissue block that contains excess embedding medium.
As the frozen tissue block advances towards the fixed blade, it gets cut into slices of desired thickness.
Carefully transfer the sliced frozen tissue sections onto positively charged glass slides. The charged glass slides electrostatically attract the frozen tissue sections, facilitating their adhesion to the slide surface.&#160;&#160;
Finally, store the sections at low temperatures until further analysis.

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3.2K 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) In this video, we demonstrate the sectioning of a frozen mouse brain tumor tissue using a cryostat. The frozen brain tissue sections so obtained are stored at low temperatures until further analysis. 

Video: Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue - Concept

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

JoVE Journal

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Biology

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...

Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue

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