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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. Sample Preparation for Laser Capture Microdissection
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	<li>Prepare solutions for staining and dehydration.
	<ol>
		<li>Place 45 mL of 80% ethanol in each of three 50 mL centrifuge tubes on ice. Label them as #1, #2, and #3. Dilute ethanol with RNase/DNase free water.</li>
		<li>Place 45 mL of 95% ethanol in a 50 mL centrifuge...

cresyl violet staining of tissue sections: a staining procedure to visualize cartilage and bones in frozen mouse embryo sections

Source: Wu, Meng, et al., <a href="https://www.jove.com/v/60503">Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis.</a> J. Vis. Exp.&#160;(2019)
This video describes cresyl violet staining procedure to visualize bones and cartilage in coronal sections of frozen mouse embryos. This staining technique helps to differentiate bones and cartilage with distinct colors from the surrounding tissues.

Cresyl Violet Staining of Tissue Sections: A Staining Procedure to Visualize Cartilage and Bones in Frozen Mouse Embryo Sections

Source: Wu, Meng, et al., Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis. J. Vis. Exp.&#160;(2019)
This ...

To visualize cartilage and bone regions in a tissue section, begin with a glass slide carrying a thin frozen mouse embryo tissue section of interest.
Briefly thaw the cryosection and immerse it in suitable ethanol concentration to remove the cryo-embedding medium. The removal step helps prevent loosening of the medium, which may otherwise affect tissue visualization during staining. Additionally, ethanol fixes the tissue, maintaining tissue morphology and integrity.
Next, pipette a cresyl violet solution onto the tissue. Cresyl violet, a positively charged, basic dye, binds to polyanionic moieties such as proteoglycans in the cartilage tissue matrix. Whereas, in mineralized tissue such as bones, it reacts with negatively charged groups such as phosphate groups in the bone matrix.
Being a metachromatic dye, cresyl violet stains different regions of the tissue in colors different from the natural dye based on its interaction with different chemical constituents within those regions.
Now, wash the tissue in a suitable ethanol concentration to remove excess stain. Immerse the tissue in increasing ethanol concentrations to replace the water from the tissue, dehydrating the tissue. Wash the tissue with xylene to displace ethanol, making the tissue transparent for microscopy.
Under a light microscope, cartilage tissue appears magenta while the bone tissue appears brown, facilitating their differentiation from each other as well as the surrounding tissue.

cresyl-violet-staining-tissue-sections-staining-procedure-to

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Tissue staining

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 &#956;m thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 &#956;m working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritic&#160;trees and dendrite spines may be reliably visualized and quantified.

We present a protocol for using the Golgi-Cox staining method in thick brain sections, in order to visualize neurons with long dendritic&#160;trees contained within single tissue samples. Two variants of this protocol are also presented that involve cresyl violet counterstaining, and the freezing of unprocessed brains for long-term storage.

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within ...

JoVE Journal

Neuroscience

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Cresyl Violet Staining of Tissue Sections: A Staining Procedure to Visualize Cartilage and Bones in Frozen Mouse Embryo Sections

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