Name: Drosophila Larval NMJ Immunohistochemistry (Video) | JoVE Journal
Uploaded: 2009-03-28T00:00:00.000+00:00
Duration: 10 min 10 s
Description: Learn immunohistochemistry for synapse development using Drosophila larvae. This protocol visualizes proteins at the neuromuscular junction to study genetic regulation of neural development.

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5 minutes

Before you start
<ol>
<li>Prepare the following solutions: PBT (0.1 % Triton X100 in 1X PBS), PBTB ( 0.2% BSA in PBT), and PBTN (2% NGS in PBTB).</li>
<li>Dissect Drosophila larvae. Please see Drosophila Larval NMJ Dissection.</li>
</ol>
Immunohistochemistry
<ol>
<li>Move the larvae to a 1.5 ml tube containing PBT. Wash the larvae twice for 15 minutes in the PBT. Note: to wash, place the 1.5 ml tube on a nutator mixer. Remove the liquid with a pippetor and replace it with fresh liquid.</li>
<li>Remove the PBT. Wash in PBTB twice for 30 minutes.</li>
<li>Remove the PBTB. Wash in PBTN twice for 15 minutes.</li>
<li>Incubate in primary antibody diluted in PBTN at room temp for 1 hour or at 4&ordm; C overnight.</li>
<li>Rinse twice with PBTB. Note: to rinse add solution and remove quickly.</li>
<li>Wash twice for 15 minutes in PBTB.</li>
<li>Wash in PBTN for 30 minutes.</li>
<li>Incubate in secondary antibody (and conjugated primary antibody if you are using one) diluted in PBTN.</li>
<li>Cover in tinfoil and incubate for 1.5 hours at room temperature.</li>
<li>Rinse twice with PBTB.</li>
<li>Wash twice for 15 minutes with PBTB.</li>
<li>Proceed to mounting.</li>
</ol>
Mounting
Note : Mount in glycerol if samples will be imaged immediately. Mount in Prolong Gold if samples will be stored before imaging (for more than a week) or if samples must be imaged multiple times. To use, place a bottle of prolong in 65&ordm;C water bath for 2-3 minutes. Take an aliquot of Prolong Gold and keep in on a heat-block at 45-50&ordm; C.
<ol>
<li>Pour stained preps in PBT in to a watch glass.</li>
<li>Put some glycerol on a clean glass slide for processing.</li>
<li>Move the animals to processing slide by picking them out of the watch glass by a corner using forceps. Ensure they are cuticle side down.</li>
<li>Remove the head and tail with a fresh razor blade on the processing glass slide. An exacto-knife with blade #16 works well for this.</li>
<li>On another slide (mounting slide), put a small drop of glycerol/prolong and spread it with clean forceps.</li>
<li>Move the dissected animals to the mounting slide by their edge, taking care not to invert them. Try to mount them in rows in the same orientation. Mount 6-8 animals per slide.</li>
<li>Drop a cover slip on by placing an edge into the glycerol/Prolong and slowly releasing it.</li>
<li>Seal the slide with nail varnish. Note [Do not image until the varnish is dry. This usually takes ten minutes. For samples mounted in prolong gold, let the slides dry for at least three hours before sealing or imaging.]</li>
</ol>

<ol><li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Properties+of+the+larval+neuromuscular+junction+in+Drosophila+Melanogaster%5BTitle%5D)+AND+%22J.+Physiol%22%5BJournal%5D)">Properties of the larval neuromuscular junction in Drosophila Melanogaster.</a> J. Physiol. 262, 189-214 (1976).</li>
<li>Johansen, J., Halpern, M. E., Johansen, K. M., Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stereotypic+morphology+of+glutamatergic+synapses+on+identified+muscle+cells+of+Drosophila+larvae%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae.</a> J Neurosci. 9, 710-725 (1989).</li>
<li>Kesheshian, H., Broadie, K., Chiba, A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Drosophila+neuromuscular+junction%3A+a+model+system+for+studying+synaptic+development+and+function%5BTitle%5D)+AND+%22Annu+Rev+Neurosci%22%5BJournal%5D)">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545-575 (1996).</li>
<li>Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptic+development%3A+insights+from+Drosophila%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">Synaptic development: insights from Drosophila.</a> Curr Opin Neurobiol. 17 (1), 35-42 (2007).</li>
</ol>

Immunohistochemistry (IHC) is vital for the study of NMJ biology because it enables visualization of the NMJ. This is accomplished by using antibodies that recognize the neuronal membrane (e.g., HRP), the presynapse (e.g., CSP, SYT), and/or the postsynapse (e.g., DLG). Signaling molecules, structural proteins, or novel proteins of interest can also be stained. Then, genes can be mutated or missexpressed, and IHC can detect perturbation of synaptic structure and/or neuronal signaling.
The NMJ o...

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drosophila larval nmj immunohistochemistry

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

This protocol describes how to perform immunohistochemical staining on dissected gsof larvae to visualize proteins involved in synapse development. A 1.7 mil tube is used throughout the procedure for primary and secondary antibody incubation, as well as for the wash steps. Larvae are then transferred on a processing slide where they're trimmed with an exacto knife for viewing specimens are moved to a drop of anti fading reagent.On a mounting slide, a cover slip is finally sealed to the slide with nail polish. Hi, I'm Jonathan Brent from the laboratory of Brian McCade in the Department of Physiology and Cellular biophysics at Columbia University College of Physicians and Surgeons. We use this procedure to study the genetic regulation of synapse formation at the neuromuscular junction and developing an node.So let's get started. Before performing immunohistochemistry on the gsof larva n MJ first properly dissect the larvae. To learn this procedure, watch the accompanying video article entitled Drosophila Larva N MJ Dissection.To begin the procedure, prepare three fresh solutions. First, make PBT Solution by adding detergent to freshly made PBS. Second, take an aliquot of bovine serum albumin and add it to a PBT solution.To make PBTB third, take an aqua normal goat serum and add it to PB TB solution to make PBTN solution. After preparing these solutions, transfer the larvae into a 1.5 mil tube containing one mil PBT. This is the first wash.Every wash involves replacing the solution in which the larvae are immersed and then incubating the larvae on a slowly rotating mutator mixer. Do not transfer larvae from tube to tube as this will damage them. This first wash is for 15 minutes at room temperature.After 15 minutes of mixing, replace the PBT with fresh PBT using a pipette and wash the larvae for another 15 minutes. When exchanging wash solutions, always be careful to avoid damaging larvae When the PBT washes are complete. Wash the larvae in PBTB for 30 minutes.Repeat this wash with fresh solution for another 30 minutes. With the PBTB wash is complete. Wash twice with PBTN for 15 minutes per wash.During the second washing step, primary antibody can be diluted in PBTN at the desired working concentration. Following the wash, incubate the larvae in this antibody mixture for one hour with gentle mixing at room temperature or overnight at four degrees Celsius. When the incubation is complete, siphon away the primary antibody solution and quickly rinse the larvae with PBTB several times to remove any remaining antibody.To prepare the larvae for the next antibody incubation, wash them once in PBTB for 15 minutes and wash twice with PBTN for 30 minutes. Dilute the secondary antibody in PBTN at the desired working concentration and incubate the larvae in this mixture for one and a half hours. At room temperature, make sure to cover the larvae to prevent light exposure and to minimize floor four bleaching.After one and a half hours, remove the secondary antibody solution and rinse the larvae several more times in PBTB. Now, wash the larvae in PBTV for 15 minutes twice. All the remaining washing and storage of the larvae should be done with minimal exposure to light, so be sure to keep them covered.Transfer the larvae from the 1.5 mil tube into PBT in a glass processing slide. The larvae are now ready for mounting to mount the stained larvae heat prolonged gold solution in a water bath at 65 degrees Celsius for two to three minutes. Then take an Eloqua and keep it on a heat block set at 50 degrees Celsius.Apply some PB TB to a processing slide. Transfer the larvae to the PB tb. Rotate the larvae so they are cuticle side down using an exacto knife with blade number 16, trim the heads and tails from the larvae.You can also use a razor for this step. Now prepare the mounting slide to a clean slide at 40 microliters of prolonged gold from the heated aliquot and spread it around with clean forceps. Using forceps.Carefully transfer six to eight of the prepared larvae from the processing slide to the mounting slide. Try to keep them in the same orientation and keep their cuticle sides down to mount the larvae. Drop a cover slip over the prolonged solution.Set it up on one edge at the periphery of the prolonged pool and carefully drop the slip to cover the samples. Seal the edges of the cover slip to the slide with your favorite color of thick, cheap nail polish. Let the slide dry for a minimum of three hours.If the slides will be imaged only once and immediately after preparation, then the prolonged gold solution may be substituted with glycerol. Then the drying time of the nail polish and glycerol is only about 10 minutes as opposed to three hours with any staining protocol. The goal is high specific signal combined with low background.This is achieved by using high quality antibodies and by thoroughly performing the blocking and washing steps. These images show wild type synapses as well as those from a fly strain expressing a mutated type two BMP receptor known as the wishful thinking or witch mutant. The neuronal membrane is stained blue with an antibody against horse radish peroxidase.Synaptic vesicles shown in red are stained with antis synaptic antibody. The post synapse shown in green is stained with antibody against discs large. These results showed that mutating the type two BMP receptor wit produces a synaptic undergrowth phenotype.This mutant was key in establishing BMPs as retrograde signals that regulate synaptic terminal buton number. We've just shown you how to perform immunohistochemistry on a esophagal larval neuromuscular junction. When doing this procedure.It's important to execute the procedure with proper timing to ensure maximum reproducibility and low background. So that's it. Thanks for watching and good luck with your experiments.

drosophila-larval-nmj-immunohistochemistry

Research

JoVE Journal

Biology

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila Larval NMJ Dissection

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures  for up to a few days1.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila Pupal Abdomen Immunohistochemistry

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

Learn immunohistochemistry for synapse development using Drosophila larvae. This protocol visualizes proteins at the neuromuscular junction to study genetic regulation of neural development.

Drosophila Larval NMJ Immunohistochemistry

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