eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review.
1. Preparation Before Tissue Collection
<ol>
	<li>Set the centrifuge to 4 oC.</li>
	<li>Fire polish a set of three glass Pasteur pipettes with decreasing diameters of approximately 1.3, 0.8, and 0.4 mm for each sample.</li>
	<li>Prepare labeled 1.7 ml tubes containing 1 ml cold Buffer A and keep the tubes on ice.</li>
	<li>Prepare an ice tray containing the brain slicing matrix, spatulas and glass plates (or inverted glass Petri dish) on which to perform the dissection. Pre-chill two or more razor blades on the glass plates and use tissues paper to dry all instruments that touch the tissue.</li>
	<li>Thaw the Enzyme solution at room temperature (RT) for 30 min before tissue collection.</li>
</ol>
2. Tissue Collection and Dissection
<ol>
	<li>Initiate the behavioral or pharmacological treatment 90 min before tissue collection. 
	NOTE: For the results shown here, acute intraperitoneal injections of 5 mg/kg methamphetamine on rats in a novel environment (test condition) and na&#239;ve rats kept in their home cages (control condition) were performed.</li>
	<li>Anesthetize the rat by placing it in a glass desiccator jar with saturated isoflurane and decapitate the rat 30 - 60 sec later using a guillotine. 
	Use scissors to remove the skin and muscle from the head and expose the skull. Use rongeurs to cut the skull, open up the foramen magnum, and remove the back part of the skull. Use rongeurs to cut along the top edges of the skull to expose the brain. Be careful not to damage the brain. Use a small spatula to gently scoop under and elevate the brain. Raise the brain and cut the nerves until the brain is free.</li>
	<li>Dissect tissue using razor blades. 
	NOTE: Obtain tissue samples using either of the following methods: (2.3.1) freshly dissected tissue; (2.3.2) frozen tissue after dissection; or (2.3.3) frozen tissue dissected from frozen whole brain. Keep the brain slicing matrix, razor blades and glass plate dry throughout the dissection and mincing processes as condensed water can lead to hypotonic lysing of cells. Use magnifying glasses to ensure accurate dissection.
	<ol>
		<li>For freshly dissected tissue, place the freshly extracted brain into a brain-slicing matrix (a rat brain-shaped metal mold with slots for the razor blades at 1 mm intervals) that has been chilled on ice. Insert two or more pre-chilled razor blades into the slots to cut coronal slices that contain the brain region(s) of interest. Place the cut slice on the chilled glass plate. 
		Note: Dissect the brain regions of interest on a chilled glass plate.</li>
		<li>For frozen tissue after dissection, dissect the brain region(s) of interest from slices of freshly extracted brain, similar to that described above in 2.3.1. Place the dissected tissue into a microtube and rapidly freeze the tissue by submerging the microtube in -40 &#186;C isopentane for 20 sec. Keep dissected tissue frozen at all times in a -80 &#186;C freezer until further processing.</li>
		<li>For frozen tissue, immediately freeze the freshly extracted brain in -40 &#186;C isopentane and store in a sealed bag at -80 &#186;C for up to 6 months.
		<ol>
			<li>On the day of dissection, place the frozen brain in a cryostat set at approximately -20 &#186;C (no warmer than -18 &#186;C) for approximately 2 hr to equilibrate the temperature. 
			NOTE: Brains can be cut easily at this temperature, while much cooler temperatures make the brain too brittle to be cut safely.</li>
			<li>Use razor blades to cut 1 - 2 mm coronal slices of the frozen brains in a cryostat. Under freezing conditions, use a blunted 12&#8212;to 16-gauge needle to obtain tissue punches from these slices. Keep dissected tissue frozen at all times until further processing.</li>
		</ol>
		</li>
		<li>Add 1 - 2 drops of Buffer A to cover the dissected tissue before mincing. Remove most of the white matter from the tissue using two razor blades to prevent the loss of neurons during the rest of the trituration process. If starting with frozen tissue, then allow the tissue to thaw on the cold glass plate for no more than 1 min before applying Buffer A solution.</li>
		<li>Mince the tissue 100 times in each orthogonal direction with a razor blade on a chilled glass plate. Hold the razor blade vertically to the glass plate when mincing. 
		NOTE: Thorough mincing is critical for all protocol steps.</li>
		<li>Use razor blades to transfer the minced tissue into microcentrifuge tubes containing 1 ml of cold Buffer A. Invert the tube 3 - 5 times to keep all minced tissue in the solution.</li>
	</ol>
	</li>
</ol>
3. Cell Dissociation
NOTE: Use gentle end over end mixing for all of the following steps. Do not use a vortex mixer and avoid air bubbles that cause cell damage.
<ol>
	<li>Centrifuge the sample tubes at 110 x g for 2 min at 4 &#186;C. Discard supernatant. Slowly add 1 ml of cold, freshly thawed Enzyme solution (containing a mix of proteolytic enzymes) down the inner wall of the microtube. Immediately suck up the entire pellet and gently pipette up and down only 4 times with a moderately large tip diameter pipette to disperse the minced tissue pellet.</li>
	<li>Invert the microtubes immediately to prevent the pellet from sticking to the bottom, and incubate the samples with end-over-end mixing for 30 min at 4 oC.</li>
	<li>After enzymatic tissue digestion, centrifuge the tubes at 960 x g for 2 min at 4 &#186;C. Remove the supernatant and add 0.6 ml of cold Buffer A. Immediately after adding Buffer A, use the same pipette tip to disperse the pellet by sucking up the entire pellet and gently pipette up and down 5 times.</li>
	<li>Mechanically triturate the digested tissue and collect in 15 ml tubes using the following steps. For each sample, use a separate set of 3 fire-polished glass Pasteur pipettes with descending diameters of approximately 1.3, 0.8, and 0.4 mm attached to latex bulbs.
	<ol>
		<li>Gently triturate each sample 10 times with the 1.3 mm glass pipette. Let the sample settle for 2 min on ice (the debris and undissociated cells will settle to the bottom). Collect the supernatant (~ 0.6 ml) and transfer it to a 15 ml conical tube (tube #1).</li>
		<li>Add 0.6 ml Buffer A solution to the remaining pellet. Triturate the sample 10 times with the 0.8 mm glass pipette. Let the sample settle for 2 min on ice. Collect the supernatant (~ 0.6 ml) and transfer it to the same 15 ml conical tube #1.</li>
		<li>Add 0.6 ml Buffer A solution to the remaining pellet. Triturate the sample 10 times with the 0.4 mm glass pipette. Let the sample settle for 2 min on ice. Collect the supernatant (~ 0.6 ml; without touching the pellet with non-dissociated cells) and transfer to the same 15 ml conical tube #1. 
		NOTE: Keep the 0.4 mm diameter pipette in the tube #1 for the following trituration steps.</li>
		<li>Repeat step 3.4.3 three more times using the smallest glass pipette (~ 0.4 mm diameter), and collect the supernatant into a separate 15 ml conical tube (tube #2).</li>
		<li>Triturate the collected cell suspensions in tubes #1 and #2 for 10 more times using the 0.4 mm glass pipette and keep them on ice.</li>
	</ol>
	</li>
</ol>
4. Cell Fixation and Permeabilization
<ol>
	<li>Prepare 4 microtubes per sample (two microtubes for cells from tube #1 and two for cells from tube #2) by adding 800 &#181;l of 100% cold ethanol (stored at -20 oC before use) into each tube and keep them on ice. Note: The final ethanol concentration will be 50%.</li>
	<li>Pipette ~ 800 &#181;l of the cell suspensions (from tube#1 and tube#2) into each of the 4 tubes containing cold ethanol and invert the tubes to mix the samples.</li>
	<li>Incubate tubes on ice for 15 min while inverting the tubes every 5 min.</li>
	<li>After fixation/permeabilization, centrifuge the tubes at 1,700 x g for 4 min at 4 &#186;C and discard the supernatant. Leave 50 &#181;l of solution to prevent cell loss. 
	NOTE: The pellets at this stage are white and stick less to the wall of the tubes than before permeabilization. Therefore, remove the supernatant carefully by touching the wall of the microtube where there is no pellet and draw up the supernatant slowly using a micropipette.</li>
</ol>
5. Cell Filtration
<ol>
	<li>Re-suspend the pellets from step 4.4 with cold PBS as follows:
	<ol>
		<li>Add 550 &#956;l of cold PBS to one of the two microtubes coming from tube #1, and use a moderately large diameter pipette tip to gently pipette the cell suspension up and down 5 times. Repeat the same for one microtube coming from tube #2.</li>
		<li>For cells from tube #1, using the same pipette, transfer the first cell suspension to the second pellet. Resuspend the second pellet by gently pipetting the combined cell suspension up and down 5 times.</li>
		<li>For cells from tube #2, resuspend and combine the pellets (i.e., third and fourth microtubes) as described in 5.1.2.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brain matrix</td>
			<td>CellPoint Scientific</td>
			<td>69-2160-1</td>
			<td>To obtain coronal brain slices</td>
		</tr>
		<tr>
			<td>Hibernate A low fluorescence</td>
			<td>Brain Bits</td>
			<td>HA-lf</td>
			<td>Buffer A&#39; in the protocol is used for processing tissue and cells from the dissociation to fixation steps</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Millipore</td>
			<td>SCR005</td>
			<td>Enzyme solution&#39; in the protocol is used for enzymatic digestion of tissue prior to trituration</td>
		</tr>
		<tr>
			<td>Cell Strainer, 40 &#181;m</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>To filter cell suspension</td>
		</tr>
		<tr>
			<td>Cell Strainer, 100 &#181;m</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td>To filter cell suspension</td>
		</tr>
		<tr>
			<td>Falcon 5 ml round-bottom polystyrene test tube with cell strainer snap cap</td>
			<td>BD Bioscience</td>
			<td>352235</td>
			<td>To filter cell suspension before passing though the flow cytometer</td>
		</tr>
		<tr>
			<td>Pasteur Pipet, Glass</td>
			<td>NIH supply</td>
			<td>6640-00-782-6008</td>
			<td>To do tissue trituration</td>
		</tr>
	</tbody>
</table>

obtaining a single-cell suspension from frozen rat brain tissue

Source: Rubio, F. J., et. al. <a href="https://app.jove.com/v/54358">Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue.</a> J. Vis. Exp. (2016)
This video demonstrates the dissociation of frozen rat brain tissue into a single-cell suspension using enzymatic digestion and mechanical trituration. The prepared cells are fixed and permeabilized with ethanol for downstream applications like flow cytometry or gene expression analysis.

Obtaining a Single-Cell Suspension from Frozen Rat Brain Tissue

Source: Rubio, F. J., et. al. Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat ...

Thaw a section of frozen rat brain tissue.
Add buffer and mince the tissue. Then, transfer it to a tube containing buffer and mix.
Centrifuge the mixture and discard the supernatant.
Add a proteolytic enzyme solution to the tissue and resuspend it using a large-diameter pipette.
Incubate the mixture to break down the extracellular matrix, loosening the neural cells.
Centrifuge the mixture and discard the supernatant.
Resuspend the tissue fragments in a chilled buffer and pipette them repeatedly to dissociate the cells.
Allow the debris and undissociated cells to settle, then transfer the single-cell suspension to a new tube.
Transfer the cells into ice-cold ethanol and mix. Incubate them to fix and permeabilize the cells.
Centrifuge the suspension and discard the supernatant.
Resuspend the neural cells in a cold buffer for further analysis.

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49 Views. Source: Rubio, F. J., et. al. Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue. J. Vis. Exp. (2016) This video demonstrates the dissociation of frozen rat brain tissue into a single-cell suspension using enzymatic digestion and mechanical trituration. The prepared cells are fixed and permeabilized with ethanol for downstream applications like flow cytometry or gene expression analysis. 

Video: Obtaining a Single-Cell Suspension from Frozen Rat Brain Tissue - Concept

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

JoVE Journal

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the animal kingdom and can be recognized as either adaptive or maladaptive responses to a wide range of stress modalities. Rodents provide a proven experimental model for translational studies addressing the integrative mechanisms of anxiety at the molecular, cellular, and circuit levels. In particular, the chronic psychosocial stress paradigm elicits maladaptive responses mimicking anxiety-/depressive-like behavioral phenotypes that are analogous between humans and rodents. While previous studies show significant effects of chronic stress on neurotransmitter contents in the brain, the effect of stress on neurotransmitter receptor levels is understudied. In this article, we present an experimental method to quantitate the neuronal surface levels of neurotransmitter receptors in mice under chronic stress, especially focusing on gamma-aminobutyric acid (GABA) receptors, which are implicated in the regulation of emotion and cognition. Using the membrane-impermeable irreversible chemical crosslinker, bissulfosuccinimidyl suberate (BS3), we show that chronic stress significantly downregulates the surface availability of GABAA receptors in the prefrontal cortex. The neuronal surface levels of GABAA receptors are the rate-limiting process for GABA neurotransmission and could, therefore, be used as a molecular marker or a proxy of the degree of anxiety-/depressive-like phenotypes in experimental animal models. This crosslinking approach is applicable to a variety of receptor systems for neurotransmitters or neuromodulators expressed in any brain region and is expected to contribute to a deeper understanding of the mechanisms underlying emotion and cognition.

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain

The BS3 chemical crosslinking assay reveals reduced cell surface GABAA receptor expression in mouse brains under chronic psychosocial stress conditions.

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the ...