Name: Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna (Video) | JoVE Journal
Uploaded: 2014-05-04T14:00:00.000+00:00
Duration: 5 min 21 s
Description: Learn whole mount immunostaining to visualize olfactory receptor neurons in Drosophila antennae. This technique reveals neuronal diversification mechanisms using confocal microscopy for neuroscience research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This study was supported by MEXT-Supported Program for the Strategic Research Foundation at Private Universities and JSPS Young Scientist B grant for H.T. We would like to thank Ohtake Norihito to edit the video clips.

1. Prepare apple plate
<ol>
	<li>Mix 12.5 g agar, 125 ml 100% commercially available apple juice, 12.5 g glucose, and 375 ml H2O. Microwave the mixture for 1 to 2 minutes and pour to the 3 cm cell culture dish. Store at 4 &#176;C.</li>
</ol>
2. Genetic cross
<ol>
	<li>Use the following representative genetic cross:</li>
</ol>
Or49a-mCD8::GFP/CyO x w1118
3. Dissection and staining protocol
<ol>
	<li>Anesthetize the fly and then cut the fly head vertically by holding it using a forceps.</li>
	<li>Carefully place the antennae containing portion on an apple plate.</li>
	<li>Cut the third segment of the antenna using fine dissection scissors.</li>
	<li>Place 90 &#181;l of the fixation solution (4% paraformaldehyde in 0.1% PBST (PBS with 0.1% Triton X-100)) to the middle of a glass bottom culture dish.</li>
	<li>Gently transfer the dissected antennae with a sharp needle directly to the fixation solution. If necessary, physically submerge the antennae into the solution using needle.</li>
	<li>Incubate for 40 minutes at room temperature (RT). Wash the antennae in 0.4% PBST (PBS with 0.4% Triton X-100), 3x 10 minutes in each, keeping them in the same dish. Use yellow tips to remove and add PBST solution. Use 90 &#181;l of the wash solution each time. 
	NOTE: Do not place the dish into a shaker during immunohistochemistry. Before removing or adding the solution into the dish, bring all the antennae into the center of the dish using the needle and carefully add or remove the solution from the edge of the dish.</li>
	<li>Block the antennae with 90 &#181;l of 5% normal horse serum in 0.1% PBST for 20 minutes at RT.</li>
	<li>After removing the blocking solution, incubate the antennae with 90&#181;l of primary antibodies in 0.1% PBST containing 5% horse serum for 48 hr at 4 &#176;C in a moistened container as described previously8.</li>
	<li>Wash the antennae 6x 10 minutes in 0.4% PBST.</li>
	<li>Incubate the antennae with 90 &#181;l of secondary antibodies in 0.1% PBST containing 5% horse serum for 48 hr at 4 &#176;C. Wash 6x 10 minutes using 0.4% PBST.</li>
	<li>To mount the antennae, remove PBST from the culture dish as much as possible and gradually introduce two different concentrations of glycerol to the antennae. First add 40% glycerol to the dish for 1 to 2 minutes; then remove this and add 80% glycerol.</li>
	<li>Carefully extract the antennae (including 80% glycerol) from the culture dish using yellow tip and place them onto a slide. Gently place a coverslip on top and seal the coverslip edges with nail polish. The antennae are now ready to be imaged by fluorescence microscopy.</li>
</ol>

<ol><li>Christensen, T. A., White, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aChristensen%2C+TA+%22Representation+of+olfactory+information+in+the+brain+In+The+Neurobiology+of+Taste+and+Smell%22">Representation of olfactory information in the brain In The Neurobiology of Taste and Smell</a>. , New York. 201-232 (2000).</li>
<li>Ache, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+a+common+strategy+for+transducing+olfactory+information%5BTitle%5D)+AND+%22Sem.+Cell+Biol%22%5BJournal%5D)">Towards a common strategy for transducing olfactory information.</a> Sem. Cell Biol. 5, 55-63 (1994).</li>
<li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Odorant-selective+genes+and+neurons+mediate+olfaction%5BTitle%5D)+AND+%22C.+elegans.+Cell%22%5BJournal%5D)">Odorant-selective genes and neurons mediate olfaction.</a> C. elegans. Cell. 13, 515-527 (1993).</li>
<li>Barth, A. L., Justice, N. J., Ngai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Asynchronous+onset+of+odorant+receptor+expression+in+the+developing+zebrafish+olfactory+system%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Asynchronous onset of odorant receptor expression in the developing zebrafish olfactory system.</a> Neuron. 16, 23-34 (1996).</li>
<li>Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+the+olfactory+system+makes+sense+of+scents%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">How the olfactory system makes sense of scents.</a> Nature. 413, 211-218 (2001).</li>
<li>Stockinger, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+circuitry+that+governs+Drosophila+male+courtship+behavior%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Neural circuitry that governs Drosophila male courtship behavior.</a> Cell. 121, 795-807 (2005).</li>
<li>Endo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+modification+of+Notch+targets+in+olfactory+receptor+neuron+diversification%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Chromatin modification of Notch targets in olfactory receptor neuron diversification.</a> Nat Neurosci. 15, 224-233 (2011).</li>
<li>Karim, M. R., Moore, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+analysis+of+Drosophila+larval+peripheral+sensory+neuron+dendrites+and+axons+using+genetic+mosaics%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.</a> J Vis Exp. , (2011).</li>
<li>Suh, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+population+of+olfactory+sensory+neurons+mediates+an+innate+avoidance+behaviour+in+Drosophila%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila.</a> Nature. 431, 854-859 (2004).</li>
<li>Sachse, S., Galizia, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+inhibition+for+temporal+and+spatial+odor+representation+in+olfactory+output+neurons%3A+A+calcium+imaging+study%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Role of inhibition for temporal and spatial odor representation in olfactory output neurons: A calcium imaging study.</a> J Neurophysiol. 87, 1106-1117 (2002).</li>
<li>Hallem, E. A., Ho, M. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+basis+of+odor+coding+in+the+Drosophila+antenna%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The molecular basis of odor coding in the Drosophila antenna.</a> Cell. 117, 965-979 (2004).</li>
<li>Vosshall, L. B., Wong, A. M., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+olfactory+sensory+map+in+the+fly+brain%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">An olfactory sensory map in the fly brain.</a> Cell. 102, 147-159 (2000).</li>
<li>Couto, A., Alenius, M., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular%2C+anatomical+and+functional+organization+of+the+Drosophila+olfactory+system%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Molecular, anatomical and functional organization of the Drosophila olfactory system.</a> Curr Biol. 15, 1535-1547 (2005).</li>
<li>Clyne, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Odorant+response+of+individual+sensilla+on+the+Drosophila+antenna%5BTitle%5D)+AND+%22Invert+Neurosci%22%5BJournal%5D)">Odorant response of individual sensilla on the Drosophila antenna.</a> Invert Neurosci. 3, 127-135 (1997).</li>
<li>Vosshall, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+spatial+map+of+olfactory+receptor+expression+in+the+Drosophila+antenna%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A spatial map of olfactory receptor expression in the Drosophila antenna.</a> Cell. 96, 725-736 (1999).</li>
<li>Wang, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-photon+calcium+imaging+reveals+an+odor-evoked+map+of+activity+in+the+fly+brain%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.</a> Cell. 112, 271-282 (2003).</li>
</ol>

The authors have nothing to disclose.

The dissection of the Drosophila antenna we describe is simple and easy to perform in a laboratory setting. To ensure a successful dissection, it is essential to utilize fine-edged scissors. While immunostaining the dissected antenna, it is important to incubate them in a moisture-filled container to avoid evaporation of the antibody solution. The dissected antenna has a tendency to float in the solution. Using 0.1% Triton in PBS during the fixation and blocking steps will facilitate submersion of the antenna in...

The olfactory system is used to distinguish between an immense variety of odor molecules and subsequently to send the resulting information to the higher brain centers. This input is used to precisely control fundamental animal behaviors, such as feeding and mating1-6. As each olfactory neuron type is associated with a specific set of odors, the diversification of olfactory receptor neurons (ORN)s is essential for proper olfactory system function7.
Drosophila genetics enables us to perform single cell level investigation involving molecular mechanisms associated with ORN development and physiological function 8-16. Whole mount immunostaining of Drosophila antennae has enabled us to understand in greater detail the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s7. Herein we provide a comprehensive description of a simple method to achieve this.

<table><tbody><tr><td>Stemi DV4 dissection microscope</td><td>Zeiss</td><td>Stemi DV4</td><td></td></tr><tr><td>Glass bottom culture dishes&amp;nbsp;</td><td>MatTek corporation</td><td>P35G-0-10-C</td><td></td></tr><tr><td>Dissection scissor</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>Rat anti-ELAV</td><td>Developmental Studies Hybridoma Bank</td><td>7E8A10</td><td>Dilution 1:200</td></tr><tr><td>Mouse anti-GFP</td><td>Invitrogen</td><td>A11122</td><td>Dilution 1:400</td></tr><tr><td>Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch Laboratories</td><td>711-225-152</td><td>Dilution 1:200</td></tr><tr><td>Donkey Anti-Rat IgG</td><td>Jackson ImmunoResearch Laboratories</td><td>712-165-150</td><td>Dilution 1:200</td></tr></tbody></table>

whole mount immunolabeling of olfactory receptor neurons in the drosophila antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Ensuring that both the dissection and fixation are performed quickly is a key factor in achieving success with this protocol. Using fine scissors and forceps is also crucial. After immunostaining, fluorescent labeled antennae were examined under a confocal microscope. We normally take 1µm sections using a 20x lens. We labeled Nba7 ORNs using Or49a-mCD8::GFP and counted the number of Nba ORNs in wild-type antenna. The mCD8-GFP reporter is cell membrane localized and so the expression seen in Figure 2 exhibits t...

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

The overall goal of the following experiment is to visualize the olfactory receptor neurons using whole mount antennae staining. First anesthetize the fly to cut its head vertically. Next, place the dissected fly head on an apple plate.Then cut the third segment of the antenna and place it to the glass bottom culture dish in order to perform immunohisto chemistry. After mounting the antennae in a slide glass, we can visualize the diverse types of olfactory receptor neurons through confocal Microscopic hi. We are working On do olfactory system.The olfactory system is used to distinguish between an immer variety of other molecules. The wide range of distinct olfactory neurons that exist within this system allows for the precise and coordinated transmission of information from target receptors to neurons within the brain. Today we'll describe the whole mount antenna staining to detect the diversification of these olfactory receptor neurons.In order to ensure a successful whole mount and antenna staining, it is critical to prepare a fresh batch of fixative immediately prior to beginning the protocol. Anesthetize the fly And then cut the fly head vertically by holding its body with forceps. Now, carefully place the dissected antenna on an apple plate.Cut the third segment of the antenna using fine dissection scissors. Place 90 Microliters fixative solution in the center of the dish. Using a sharp needle, gently transfer the dissected antenna directly to the Fixative solution.Now allow the fixation to proceed undisturbed for 40 minutes at room temperature to end the fixation. Wash the antennae three times with PBST for 10 minutes per wash during removal of the washing solution. First, bring all the antennae to the center of the dish using a needle.Then remove the solution from the side of the dish. Agitation during incubation is Unnecessary. To prepare for Staining, remove the last wash of PBST and block the fixed antennae by filling the chamber with 90 microliters of 5%horse serum in 0.1%PBST replace the blocking solution with primary antibodies diluted in a mixture of 5%horse serum.In PBST, the primary antibodies used are mouse anti GFP one in 400 and rat anti eof, diluted to a concentration of one in 200 to prevent the antennae samples from drying out. Place a moist tissue into the lid of the culture dish and wrap it with the paraform. Then place the culture dish in a plastic container containing moist tissues.Incubate the antennae in the primary antibodies for 48 hours at four degrees Celsius after 48 hours. Clear the unbound primary antibodies with six washes in PBST for 10 minutes per wash. Similar to the previously described fixative removal procedure.After an hour of PBST washes, add secondary antibody solution diluted in a mixture of 5%horse serum In PBST in this example, the antibodies are SI two anti rabbit IgG and SI three anti rat IgG. Cover the container with aluminum foil from this point forward to prevent fluoro four. Photobleaching next incubate the antenna in the secondary antibody for 48 hours at four degrees Celsius.At this point, the antenna is now ready for mounting to mount the antenna. Remove as much of the PBST from the culture dish as possible, and gradually begin to introduce glycerol to the antennae. Subsequently, add 40%glycerol to the dish for one to two minutes, then remove it and add 80%glycerol and leave it for five minutes.Fluorescent staining revealed Elof positive neurons and the targeted olfactory neurons. By combining this procedure With other similar methods, including the generation olfactory neuron genetic mosaics, we hope to initiate the mechanical controlling olfactory neuron de diversification and answer other important scientific questions.

whole-mount-immunolabeling-olfactory-receptor-neurons-drosophila

Research

JoVE Journal

Neuroscience

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

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

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

We describe here methods for inducing and analyzing olfactory experience-dependent remodeling of antennal lobe synaptic glomeruli in the Drosophila juvenile brain.

Experience-Dependent Remodeling of Juvenile Brain Olfactory Sensory Neuron Synaptic Connectivity in an Early-Life Critical Period

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

This article describes an efficient imaginal disc dissection and antibody staining protocol that is economical with reagents and facilitates handling of imaginal discs in small numbers.

Small Scale Dissection and Antibody Staining of Eye-Antennal and Wing Imaginal Discs from Drosophila melanogaster

Learn whole mount immunostaining to visualize olfactory receptor neurons in Drosophila antennae. This technique reveals neuronal diversification mechanisms using confocal microscopy for neuroscience research.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Explore More Videos