eoe sub articles

EoE Sub Articles

1. Flash freezing and thorax bisection
<ol>
	<li>Before beginning the bisections, fill a Dewar flask with liquid nitrogen wearing proper cryo-protective gloves and safety glasses. Obtain a blade breaker, feather blades, one pair of fine forceps, ice, ice-cold 1x PBS, and cryogenic tweezers.</li>
	<li>Prepare an ice bucket to keep 1x PBS ice cold.</li>
	<li>Use the blade breaker to grab the feather blade at an angle and bend the blade in order to break off a small piece. The blade breaker can then lock the blade in position for use as a small scalpel. 
	NOTE: One blade should last for all groups. Switch if the blade breaks or becomes dull.</li>
	<li>Add a clean pipette tip to the P200 and remove 1/5th of the tip to transport samples.</li>
	<li>Prepare a new microcentrifuge tube for each group and add 200 &#956;L of 1x PBS to each tube. This second tube will be used to collect the final DLM preps.</li>
	<li>Remove all 1x PBS from tubes using a Pasteur pipette.</li>
	<li>Wearing proper protective equipment, submerge the tube into the liquid nitrogen flask for 10 s with the cryogenic tweezers. 
	NOTE: The tubes should be closed tightly to keep the tube from exploding.</li>
	<li>Remove the tube from the liquid nitrogen and add approximately 300 &#956;L of ice-cold 1x PBS to the samples with a Pasteur pipette. Keep the samples on ice.</li>
	<li>Add ice-cold 1x PBS to the 10 cm dissection dish coated with silicone elastomer and dispense the first thorax with the modified 200 &#956;L pipette.</li>
	<li>Place the thorax ventral side up. In one hand use a dull pair of forceps to position the thorax and in the other use a fine pair of forceps to remove some of the thoracic ganglion to expose the midline of the thorax.</li>
	<li>Use the midline of the thorax as a guide to make a shallow cut through 1/3rd of the thorax with the blade.</li>
	<li>Remove the blade from the thorax and position the thorax at a 45&#176; angle with the blunt forceps. Reinsert the blade and cut straight down the midline of the thorax. This will result in two hemithoraces.</li>
	<li>Take one hemithorax at a time and remove the excess tissue under DLM muscle fiber F (Figure 1B), the most ventral fiber. Use the blade to carefully make one or two cuts to remove the excess tissue without damaging the DLMs.</li>
	<li>Once isolated, transfer the hemithorax to the correct tube with 1x PBS.</li>
	<li>Repeat steps 1.6&#8210;1.14 until 10 dissected hemithoraces per group are made.</li>
</ol>
2. Structural staining
<ol>
	<li>After bisecting the thorax samples, place the tissue in blocking buffer (1x PBS with 0.1% normal goat serum, and 0.2% Triton X-100 at pH 7.4) to permeabilize the tissue and prevent non-specific staining. Use a Pasteur pipette to remove excess 1x PBS and add 1.5 mL of blocking buffer to each tube. Block tissues for at least 1 h at 4 &#176;C.</li>
	<li>Prepare the samples for structural staining using a fluorescently conjugated antibody, horseradish peroxidase 488 (anti-HRP-488) at a dilution of 1:200 and Phalloidin-647 at a dilution of 1:1000 in blocking buffer to stain motor neurons and muscle tissue, respectively. Make enough stain to have 150 &#956;L per tube. Store the stain at 4 &#176;C covered in foil or in a dark box until ready for staining.</li>
	<li>After blocking, remove the excess blocking buffer with a glass Pasteur pipette.</li>
	<li>Before dispensing the structural stain, vortex the stain. Add 150 &#956;L of the stain to each tube. Place the samples in a dark box on the rotator at room temperature for 2 h.</li>
	<li>Remove the stain and wash the tissues four times in 1.5 mL of room temp 1x PBS with 0.3% Triton X-100 for 5 min on the rotator in a dark box. The samples are now ready to mount to a slide.</li>
</ol>
3. Mounting tissue
<ol>
	<li>After washing samples in PBST, prepare a microscope slide to mount tissue for staining. Prepare additional supplies including glass cover slips, a P200 pipette, 200 &#956;L pipette tips, scissors, clear reinforcements, straight edge forceps, anti-fade fluorescent mounting media, nail polish, and a dark box to cover the slides.</li>
	<li>Label the slide to identify the samples and clean it with Kim wipes to remove any smudges.</li>
	<li>To ensure the cover slip does not damage the hemithorax samples, build a &#34;bridge&#34; using reinforcement labels. Cut one reinforcement label in half and place each half approximately 15 mm apart. This distance must be smaller than the width of the cover slip. Repeat this step four times to complete a &#34;bridge&#34; that is 5 labels high.</li>
	<li>Take the P200 pipette and modify a tip by cutting off 1/5th of the tip to transfer the samples to the slide. Samples should be transferred onto the slide in the center of the bridge.</li>
	<li>Take the edge of a lab wipe and remove any excess PBST. Using forceps, arrange the DLMs such that all samples are facing muscle side up and cuticle side down.</li>
	<li>Using a standard P200 pipette tip, apply 70 &#956;L of mounting media to the slide, avoiding air bubbles. Dispense the media in a circular pattern inside the reinforcements starting from the outside into the center.</li>
	<li>Place a cover slip over the reinforcements.</li>
	<li>Use nail polish to coat the outside edges around the perimeter of the coverslip. Apply generously to form a complete seal of the tissue.</li>
	<li>Place the slide on a flat surface in the dark, allowing at least 10 min to dry and prevent photo-bleaching or loss of fluorescence. Slides may now be used for imaging immediately, or otherwise stored in a slide folder at -20 &#176;C for later viewing.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>32% Formaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td>Tissue preservation</td>
		</tr>
		<tr>
			<td>BenchRocker</td>
			<td>Genesee Scientific</td>
			<td>31-302</td>
			<td>Rotating samples during staining</td>
		</tr>
		<tr>
			<td>Blade Breaker</td>
			<td>Fine Science Tools</td>
			<td>10053-09</td>
			<td>Used for holding feather blade</td>
		</tr>
		<tr>
			<td>cover slips</td>
			<td>Fisher Scientific</td>
			<td>12548A</td>
			<td>For mounting tissue</td>
		</tr>
		<tr>
			<td>cryogenic gloves</td>
			<td>VWR</td>
			<td>97008-198</td>
			<td>protect hands from liquid nitrogen</td>
		</tr>
		<tr>
			<td>cryogenic tweezers</td>
			<td>VWR</td>
			<td>82027-432</td>
			<td>Hold 2.0 mL tube in liquid nitrogen</td>
		</tr>
		<tr>
			<td>dewar flask-1900 mL</td>
			<td>Thomas Scientific</td>
			<td>5028M54</td>
			<td>Hold liquid nitrogen</td>
		</tr>
		<tr>
			<td>Feather Blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>72002-01</td>
			<td>Scalpel Blades</td>
		</tr>
		<tr>
			<td>Fine Forecps x 2</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>One fine pair for Clearing midline of thorax. The other pair can be dulled using a sharpening stone.</td>
		</tr>
		<tr>
			<td>FITC-conjugated anti HRP</td>
			<td>Jackson Laboratories</td>
			<td>123-545-021</td>
			<td>Stains Motor Neurons. Used at 1:100 concentration</td>
		</tr>
		<tr>
			<td>freezer box (Black)</td>
			<td>Fisher Scientific</td>
			<td>14100F</td>
			<td>Protects samples from light</td>
		</tr>
		<tr>
			<td>glass pasteur pipettes</td>
			<td>VWR</td>
			<td>14637-010</td>
			<td>Used to transfer samples</td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td>Fisher Scientific</td>
			<td>12550143</td>
			<td>For mounting tissue</td>
		</tr>
		<tr>
			<td>mounting media (vectashield) anti- fade</td>
			<td>VWR</td>
			<td>101098-042</td>
			<td>Mounting media retains fluorescent signaling</td>
		</tr>
		<tr>
			<td>nail polish</td>
			<td>Electron Microscopy Sciences</td>
			<td>72180</td>
			<td>Seals microscope slides</td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Fisher Scientific</td>
			<td>PCN5000</td>
			<td>Prevents non-specific binding of antibodies</td>
		</tr>
		<tr>
			<td>paint brush</td>
			<td>Genesee Scientific</td>
			<td>59-204</td>
			<td>Transferring flies</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>10-010-023</td>
			<td>Saline solution for dissecting and staining</td>
		</tr>
		<tr>
			<td>Phalloidin 647</td>
			<td>Abcam</td>
			<td>AB176759</td>
			<td>Stains F-Actin in muscle Tissue. Used at 1:1000 concentration</td>
		</tr>
		<tr>
			<td>plastic petri dish (100 mm)</td>
			<td>VWR</td>
			<td>25373-100</td>
			<td>Dissection dish</td>
		</tr>
		<tr>
			<td>reinforcement labels</td>
			<td>W.B. Mason</td>
			<td>AVE05722</td>
			<td>Provides support for glass coverslip over the mounted tissue</td>
		</tr>
		<tr>
			<td>sharpening block</td>
			<td>Grainger</td>
			<td>1RDF5</td>
			<td>Keeping fine forceps sharp and also dulling separate pair</td>
		</tr>
		<tr>
			<td>slide folder</td>
			<td>VWR</td>
			<td>10126-326</td>
			<td>Sample storage</td>
		</tr>
		<tr>
			<td>standard office scissors</td>
			<td>W.B. Mason</td>
			<td>ACM40618</td>
			<td>Cutting reinforcement labels</td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Electron Microscopy Sciences</td>
			<td>24236-10</td>
			<td>Coating for dissection dish</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Electron Microscopy Sciences</td>
			<td>22140</td>
			<td>Helps to permeabilize tissue</td>
		</tr>
		<tr>
			<td>Vannas Disssection Sissors</td>
			<td>Fine Science Tools</td>
			<td>1500-00</td>
			<td>Ued for removing fly legs and making an incision on thorax</td>
		</tr>
	</tbody>
</table>

assessing the structural integrity of the dorsal longitudinal muscle neuromuscular junctions in drosophila

Source: Sidisky, J. M., et al. <a href="https://app.jove.com/v/61363">Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration.</a> J. Vis. Exp. (2020).
This video demonstrates a protocol for assessing the structural integrity of neuromuscular junctions in the dorsal longitudinal muscles of Drosophila. The procedure involves staining neurons and muscles with two distinct antibodies and comparing fluorescence patterns between mutant and wild-type flies. A reduction in neuronal staining in mutant flies indicates neurodegeneration, while the muscle structure remains intact.

<img alt="Figure 1" src="/files/ftp_upload/22991/22991_Figure1.jpg" /> 
Figure 1: Progressive denervation of DLM synapses in a Drosophila model of ALS. (A) The generation of ALS transgenic flies expressing a human mutant form of Tar-Binding Protein of 43 kDa (TDP-43) are shown in the schematic. (B) The illustration depicts the shape and orientation of a hemithorax in an adult Drosophila. Using the protocol, we can o...

Assessing the Structural Integrity of the Dorsal Longitudinal Muscle Neuromuscular Junctions in Drosophila

1. Flash freezing and thorax bisection

	Before beginning the bisections, fill a Dewar flask with liquid nitrogen wearing proper cryo-protective gloves ...

Begin with chemically fixed wild-type and transgenic Drosophila thoraces in a buffer.
In transgenic thoraces, the dorsal longitudinal muscles&#39; neuromuscular junctions contain neurons that express a mutant protein inducing neurodegeneration.
Remove the buffer and flash-freeze the thoraces to prevent tissue damage during bisection.
Add fresh buffer and transfer the thoraces to dissection dishes.
Position a thorax ventral side up and remove the nerve cell clusters to expose the midline.
Bisect along the midline to obtain hemithoraces.
Remove excess tissue.
Incubate the hemithoraces in a blocking buffer to permeabilize the membranes and prevent non-specific antibody binding.
Add fluorophore-conjugated antibodies targeting a glycoprotein expressed in NMJ neurons and muscle actin filaments.
Remove unbound antibodies.
Arrange the hemithoraces on a bridge slide, add mounting media, and mount the tissues.
Visualize under a microscope.
Compared to the wild-type, the mutants show reduced neuronal staining, indicating neurodegeneration, while the muscles exhibit intact fluorescence.

assessing-structural-integrity-dorsal-longitudinal-muscle

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neuromuscular Disorders

54 Aufrufe. Quelle: Sidisky, J. M., et al. Visualisierung der synaptischen Degeneration bei adulten Drosophila in Verbindung mit Neurodegeneration. J. Vis. Exp. (2020). Dieses Video zeigt ein Protokoll zur Beurteilung der strukturellen Integrität neuromuskulärer Verbindungen in den dorsalen Längsmuskeln von Drosophila. Bei dem Verfahren werden Neuronen und Muskeln mit zwei unterschiedlichen Antikörpern gefärbt und die Fluoreszenzmuster zwischen mutierten und Wildtyp-Fliegen verglichen. Eine Verringeru...

Serie: Beurteilung der strukturellen Integrität der neuromuskulären Verbindungen des dorsalen Längsmuskels bei Drosophila - Konzept

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

Eine automatisierte rasche Iterative Negative Geotaxis Assay für die Analyse von Erwachsenen Klettern Verhalten in einem Drosophila -Modell der Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

Forschung

JoVE Journal

Verhalten

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Massen Histologie zu Quantifizieren Neurodegenerative Erkrankungen in<em&gt; Drosophila</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Neurowissenschaften

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuskuläre Synapse Messfunktionalität

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Assessing the Structural Integrity of the Dorsal Longitudinal Muscle Neuromuscular Junctions in Drosophila

Transkript

Assessing the Structural Integrity of the Dorsal Longitudinal Muscle Neuromuscular Junctions in Drosophila