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EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue Preparation
<ol>
	<li>Order timed pregnant rats from the vendor.</li>
	<li>Follow timed pregnant rats by observing their growing abdomens in the weeks after their arrival and subsequently looking for pups on the expected delivery date by inspecting the cage every 2 hours until delivery begins.</li>
	<li>Remove pups with a gloved hand by their tail before their first feed for unprimed pups (no white milk belly is apparent when viewing the abdomen) or after the first feed for primed pups (at which point a white stomach will be visible on their abdomen) as described in the timeline (Figure 1). 
	NOTE: The first colostrum feed is termed as priming the pup; thus, a pup is unprimed until the first feed, after which they are colostrum-primed.</li>
	<li>Quickly decapitate the unanesthetized pup using sharp, clean surgical scissors.</li>
	<li>Remove the brain by cutting the skin down the midline and top surface of the skull to the nose. Then, using forceps, gently pry away the bone to expose the brain (Figure 2A) and localize the bregma, marking it with a pen as the bone plates are removed (Figure 2B).</li>
	<li>Rapidly place the whole brain in a polymethyl methacrylate brain mold at room temperature for coronal slicing at room temperature (Figure 2C).</li>
	<li>Without delay, make 500 &#956;m-thick slices using a fresh razor blade. Lay the slices rostral to caudal in a Petri dish to maintain the orientation of sections (Figure 3).</li>
	<li>Quickly add artificial cerebrospinal fluid (ACSF; 1.0 mM potassium dihydrogen phosphate, (KH2PO4), 26 mM sodium bicarbonate (NaHCO3), 118.6 mM Sodium chloride (NaCl), 3.0 mM potassium chloride (KCl), 203.3 mM magnesium chloride (MgCl2-6H2O)) without glucose and incubate the slices for 60 min at 28-30 &#176;C, constantly stirring on an orbital shaker to metabolically and differentially challenge the unprimed versus colostrum-primed tissues.</li>
	<li>Identify the brain nuclei that are required to punch using a brain atlas and anatomic landmarks on the tissue section. Place this slice with the nuclei of interest in a Petri dish and move it to the dissecting microscope.</li>
	<li>Once visualized, quickly punch out 4 of 6 different nuclei using a coring tool, selecting the size to best punch the nucleus in question and consistently between samples (Figure 3). 
	NOTE: The remaining brain slice will now have a hole where brain tissue was removed. In this study, we excised the following nuclei using the below coordinates. All anterior/posterior (A/P) coordinates are from Bregma (except nucleus of the solitary tract (NTS), which is with reference to the Calamus Scriptorius). All dorsal/ventral (D/V) coordinates are from the surface of the cortex (except NTS, which is from the surface of the medulla). The following coordinates include A/P, medial/lateral (M/L), and D/V in mm: 1) solitary tract nucleus (NTS, A/P, 0.4 to 0.8; L, &#177; 0.2; D/V, 0.3 (from the surface of the medulla), 2) paraventricular nucleus (PVN, -0.8; &#177; 0.2; 0), 3) supra-optic nucleus (SON, -1.1; &#177; 1.4; 4.3), 4) cortex (CX, partial cortex area 1, -2.8; &#177; 1.5; 0.6), 5) striatum nuclei (STR, -0.0; &#177; 1.6; 1.8), and 6) medial preoptic nucleus (MPO, -0.6; &#177; 0.2; 4.2).</li>
	<li>Rapidly immerse the punched nuclei in 0.06 mL of ice-cold protein extraction buffer containing protease inhibitors and phosphatase inhibitors for 60 minutes.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Pregnant Sprague-Dawley rats</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Punch device</td>
			<td>WellTech Rapid Core or Harris Uni-Core</td>
			<td>0.35, 0.50, 0.75, 1.0, 1.20, 1.50</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of oxytocin receptor-rich nuclei from a neonatal rat brain

Source: Klein, B. Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58341">Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation of oxytocin receptor-rich nuclei from the neonatal rat brain. Coronal slicing, followed by identification and precise punching of oxytocin receptor-rich brain nuclei, enables the effective extraction of oxytocin receptor-rich nuclei for further analysis.

<img alt="Figure 1" src="/files/ftp_upload/23205/23205_Figure1.jpg" /> 
Figure 1: Schematic showing the protocol timeline.
<img alt="Figure 2" src="/files/ftp_upload/23205/23205_Figure2.jpg" /> 
Figure 2: Dissected neonatal rat brain. (A) The dissected brain is shown with bones removed from rostral to bregma. (B) The brain is shown after a line has been drawn at bregma, after which the remaining bone plates were removed. (C) The brain is shown in a polymethyl methacrylate brain mold just before being sliced with a razor blade.
<img alt="Figure 3" src="/files/ftp_upload/23205/23205_Figure3.jpg" /> 
Figure 3: Brain slices arranged from rostral to caudal with punches (red circles). Abbreviations: CX: cortex, parietal lobe; MPO: medial preoptic area; NTS: nucleus tractus solitaries; PVN: paraventricular nucleus; SON: supraoptic nucleus; and STR: striatum.

Isolation of Oxytocin Receptor-Rich Nuclei from a Neonatal Rat Brain

Source: Klein, B. Y. et al., Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After ...

The brain&#39;s oxytocin receptor-rich nuclei are clusters of neurons that express oxytocin receptors, which interact with oxytocin, a neuropeptide.
To isolate oxytocin receptor-rich nuclei, begin with a neonatal rat brain.
Place the brain in a mold with a slicing guide. Slice the entire brain to expose different regions with nuclei.
Transfer these slices to a dish and immobilize them.
Add artificial cerebrospinal fluid, or aCSF, to maintain neuronal viability and incubate under gentle rotation.
Using a published brain atlas as a reference, identify anatomical landmarks to locate oxytocin-receptor-rich nuclei within the slices.
Transfer an identified brain slice of interest to a new plate.
Under a microscope, locate the nuclei within the slice and punch it.
Repeat the process for other identified sections to isolate the oxytocin-rich nuclei.
Transfer the isolated nuclei to a tube containing ice-cold protein extraction buffer supplemented with protease and phosphatase inhibitors to preserve the oxytocin receptors.

isolation-of-oxytocin-receptor-rich-nuclei-from-a-neonatal-rat-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Developmental and Pediatric Disorders

49 Views. Source: Klein, B. Y. et al., Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding. J. Vis. Exp. (2018) This video demonstrates the isolation of oxytocin receptor-rich nuclei from the neonatal rat brain. Coronal slicing, followed by identification and precise punching of oxytocin receptor-rich brain nuclei, enables the effective extraction of oxytocin receptor-rich nuclei for further analysis. 

Video: Isolation of Oxytocin Receptor-Rich Nuclei from a Neonatal Rat Brain - Concept

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused on bulk approaches that led to the molecular classification of brain tumors. Over the last five years, single cell sequencing approaches have highlighted several important features of gliomas. The majority of these studies have utilized fresh brain tumor specimens to isolate single cells using flow cytometry or antibody-based separation methods. Moving forward, the use of fresh-frozen tissue samples from biobanks will provide greater flexibility to single cell applications. Furthermore, as the single-cell field advances, the next challenge will be to generate multi-omics data from either a single cell or the same sample preparation to better unravel tumor complexity. Therefore, simple and flexible protocols that allow data generation for various methods such as single-nucleus RNA sequencing (snRNA-seq) and single nucleus Assay for Transposase-Accessible Chromatin with high-throughput sequencing (snATAC-seq) will be important for the field.
Recent advances in the single cell field coupled with accessible microfluidic instruments such as the 10x genomics platform have facilitated single cell applications in research laboratories. To study brain tumor heterogeneity, we developed an enhanced protocol for the isolation of single nuclei from fresh frozen gliomas. This protocol merges existing single cell protocols and combines a homogenization step followed by filtration and buffer mediated gradient centrifugation. The resulting samples are pure single nuclei suspensions that can be used to generate single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

JoVE Journal

Cancer Research

Gentle Isolation of Nuclei from the Brain Tissue of Adult African Turquoise Killifish, a Naturally Short-Lived Model for Aging Research

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we demonstrate a protocol to generate single-nucleus RNA sequencing (snRNA-seq) libraries from the brains of the naturally short-lived vertebrate African turquoise killifish Nothobranchius furzeri. The African turquoise killifish has a lifespan of 4-6 months and can be housed in a cost-effective manner, thus reducing cost and time barriers to study vertebrate brain aging. However, tailored protocols are needed to isolate nuclei of sufficient quality for downstream single-cell experiments from the brain of young and aged fish. Here, we demonstrate an empirically optimized protocol for the isolation of high-quality nuclei from the brain of adult African turquoise killifish, a critical step in the generation of high-quality single nuclei omic libraries. Furthermore, we show that the steps to reduce contaminating background RNA are important to clearly distinguish cell types. In summary, this protocol demonstrates the feasibility of studying brain aging in non-traditional vertebrate model organisms.

Here we present a protocol to isolate nuclei from the brains of the short-lived vertebrate model Nothobranchius furzeri for downstream applications such as single-nucleus RNA sequencing or single-nucleus assay for transposase-accessible chromatin with sequencing (ATAC-seq).

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we ...

High-Quality Sample Preparation for Multiome 10X Genomics Analysis

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. 
Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. 
The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions.
This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.

Author Spotlight: Advancing Biomedical Research Through Single Cell Analysis 

The success of single-cell/single-nuclei transcriptomics and multi-omics largely depends on the quality of cells/nuclei. Therefore, isolating cells/nuclei from tissue and their purification must be highly standardized. This protocol describes the preparation of nuclei from the brain and bone marrow for downstream single-nuclei multiome assay.

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of ...

Isolation of Oxytocin Receptor-Rich Nuclei from a Neonatal Rat Brain

Transcript

Isolation of Oxytocin Receptor-Rich Nuclei from a Neonatal Rat Brain

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Gentle Isolation of Nuclei from the Brain Tissue of Adult African Turquoise Killifish, a Naturally Short-Lived Model for Aging Research

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays