Name: quadruple immunostaining of the olfactory bulb for visualization of olfactory sensory axon molecular identity codes
Uploaded: 2017-06-05T15:00:00
Description: Watch this Scientific Journal Video about Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes at JoVE.com

This work was supported by the Mitsubishi Foundation, the Takeda Science Foundation, JST PRESTO and JSPS KAKENHI Grant Number 16H06144.

All experimental procedures were performed with the approval of the animal experiment ethics committee at the University of Tokyo and according to the University of Tokyo guidelines for the care and use of laboratory animals.
1. Preparation of Solutions
<ol>
	<li>Prepare 0.01 M Phosphate-Buffered Saline (PBS): add a PBS tablet (0.14 M NaCl, 0.0027 M KCl, 0.010 M PO43-, pH 7.4) to 1 L distilled water and stir at RT until it is completely dissolved.</li>
	<li>Prep...

<ol>
	<li>Mori, K., Sakano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+is+the+olfactory+map+formed+and+interpreted+in+the+mammalian+brain?.">How is the olfactory map formed and interpreted in the mammalian brain?.</a> Annu Rev Neurosci. 34, 467-499 (2011).</li><li>Takeuchi, H., Sakano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+map+formation+in+the+mouse+olfactory+system.">Neural map formation in the mouse olfactory system.</a> Cell Mol Life Sci. 71, 3049-3057 (2014).</li><li>Mombaerts, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+an+olfactory+sensory+map.">Visualizing an olfactory sensory map.</a> Cell. 87, 675-686 (1996).</li><li>Feinstein, P., Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+contextual+model+for+axonal+sorting+into+glomeruli+in+the+mouse+olfactory+system.">A contextual model for axonal sorting into glomeruli in the mouse olfactory system.</a> Cell. 117, 817-831 (2004).</li><li>Ishii, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoallelic+expression+of+the+odourant+receptor+gene+and+axonal+projection+of+olfactory+sensory+neurones.">Monoallelic expression of the odourant receptor gene and axonal projection of olfactory sensory neurones.</a> Genes Cell. 6, 71-78 (2001).</li><li>Nakashima, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agonist-independent+GPCR+activity+regulates+anterior-posterior+targeting+of+olfactory+sensory+neurons.">Agonist-independent GPCR activity regulates anterior-posterior targeting of olfactory sensory neurons.</a> Cell. 154, 1314-1325 (2013).</li><li>Serizawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuronal+identity+code+for+the+odorant+receptor-specific+and+activity-dependent+axon+sorting.">A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting.</a> Cell. 127, 1057-1069 (2006).</li><li>Kaneko-Goto, T., Yoshihara, S., Miyazaki, H., Yoshihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BIG-2+mediates+olfactory+axon+convergence+to+target+glomeruli.">BIG-2 mediates olfactory axon convergence to target glomeruli.</a> Neuron. 57, 834-846 (2008).</li><li>Ihara, N., Nakashima, A., Hoshina, N., Ikegaya, Y., Takeuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+axon-sorting+molecules+in+mouse+olfactory+sensory+neurons.">Differential expression of axon-sorting molecules in mouse olfactory sensory neurons.</a> Eur J Neuro. 44, 1998-2003 (2016).</li><li>Williams, E. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delta+Protocadherin+10+is+Regulated+by+Activity+in+the+Mouse+Main+Olfactory+System.">Delta Protocadherin 10 is Regulated by Activity in the Mouse Main Olfactory System.</a> Front Neural Circuits. 5, 9 (2011).</li><li>Brunet, L. J., Gold, G. H., Ngai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anosmia+caused+by+a+targeted+disruption+of+the+mouse+olfactory+cyclic+nucleotide-gated+cation+channel.">General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel.</a> Neuron. 17, 681-693 (1996).</li></ol>

Quadruple immunostaining of parasagittal OB sections enabled the visualization and quantification of the expression levels of as many as four axon-sorting molecules simultaneously in a larger number of glomeruli. By analyzing these multivariable data with PCA, the characteristics for the expression of those molecules can be speculated.
For successful staining, the tissue sample preparation is critically important. Some protocols suggest that tissues should be post-fixed with 4% PFA and treated...

During development, neurons are precisely connected with each other to form proper neural circuits, which is critical for the normal brain function. Since aberrant neural circuits in the brain are thought to be the cause of mental disorders such as autism and schizophrenia, understanding the mechanisms of neural circuit formation is one of the major challenges in the field of neuroscience.
In the mouse olfactory system, each Olfactory Sensory Neuron (OSN) in the Olfactory Epithelium (OE) expresses only one functional Olfactory Receptor (OR) gene and OSNs expressing the same OR converge their axons to a specific pair of glomeruli at stereotyped locations in the Olfactory Bulb (OB)1,2. The mouse olfactory system is an excellent model system for studying the molecular mechanisms of neural circuit formation because researchers can utilize the OR expression to identify a specific subtype of OSNs and visualize the projection sites of OSN axons as clear glomerular structures. A remarkable feature of OSN projection is that ORs play instructive roles in projecting OSN axons to the OB3,4,5,6. More specifically, after OSN axons are guided to approximate target regions, they are segregated to form glomerulus in an OR-dependent manner. Previous studies have shown that OR molecules control the expression of axon-sorting molecules, which regulate glomerular segregation7,8. Moreover, accumulating evidence suggests that OR molecules generate the neuronal identity code by a unique combination of axon-sorting molecules9. Thus, to understand the mechanism of OR-dependent glomerular segregation, it is necessary to characterize the expression profiles of axon-sorting molecules in OSNs.
Fluorescent immunostaining is a common method to visualize the expression of specific genes. Since proteins of axon-sorting molecules are predominantly localized to OSN axons, researchers need to use OB sections to characterize their expression patterns in OSNs. Coronal sectioning of the OB has been routinely used for immunostaining. However, this preparation loses the topographic information along the anterior-posterior axis in the same OB section. We therefore developed a parasagittal preparation of the medial side of the OB, which can mount as many surrounding glomeruli as possible on the same OB section. Combined with immunostaining using multiple antibodies, this preparation allows the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.
Furthermore, an immunohistochemical staining method has been presented without post-fixation with PFA and sucrose treatment. This method allows researchers to obtain enough high-quality staining data for multivariable data analysis. The protocols presented here will provide details of powerful methods for researchers who study the olfactory neural circuit formation.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Phosphate Buffered Saline (PBS) Tablets, pH7.4</td>
			<td style="width:151px;">TAKARA BIO</td>
			<td style="width:119px;">T9181</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Skim Milk</td>
			<td style="width:151px;">nacalai tesque</td>
			<td style="width:119px;">31149-75</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">goat anti-Sema7A antibody</td>
			<td style="width:151px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2068</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">rat anti-OLPC antibody</td>
			<td style="width:151px;">Merck Millipore</td>
			<td style="width:119px;">MABT20</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">mouse anti-VGLUT2 antibody</td>
			<td style="width:151px;">Merck Millipore</td>
			<td style="width:119px;">MAB5504</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">goat anti-BIG-2 antibody</td>
			<td style="width:151px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2205</td>
			<td></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:277px;">gunea pig anti-Kirrel2 antibody</td>
			<td style="width:151px;">Operon Biotechnologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Anti-Kirrel2 antibodies were generated by immunizing guinea pigs with KLH-conjugated synthetic peptides (644-673aa): CRLYRARAGYLTTPHPRAFTSYMKPTSFGP</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">donkey anti-mouse Alexa Fluor 405</td>
			<td style="width:151px;">Abcam</td>
			<td style="width:119px;">ab175658</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">donkey anti-goat Alexa Fluor 488&#160;</td>
			<td style="width:151px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">705-545-003</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">donkey anti-guinea pig Alexa Fluor 555</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A21432</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">donkey anti-rat Alexa Fluor 647</td>
			<td style="width:151px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">712-605-153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Paraformaldehyde</td>
			<td style="width:151px;">Wako</td>
			<td style="width:119px;">162-16065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">MAS coated slide glasses</td>
			<td style="width:151px;">MATSUNAMI</td>
			<td style="width:119px;">MAS-01</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">forceps</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:119px;">11253-27</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">Vannas Spring Scissors</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">dissecting scissors</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">fluorescent microscope</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">BZ-X700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DAPI filter cube</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">OP-87762</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">GFP filter cube</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">OP-87763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">TRITC filter cube</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">OP-87764</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Cy5 filter cube</td>
			<td style="width:151px;">KEYENCE</td>
			<td style="width:119px;">OP-87766</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">filter paper</td>
			<td style="width:151px;">ADVANTEC</td>
			<td style="width:119px;">00011185</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">O.C.T compound</td>
			<td style="width:151px;">Sakura Finetek</td>
			<td style="width:119px;">M71484</td>
			<td></td>
		</tr>
	</tbody>
</table>

quadruple immunostaining of the olfactory bulb for visualization of olfactory sensory axon molecular identity codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

The olfactory glomerular map is formed by initial global targeting and subsequent glomerular segregation of OSN axons1,2. Glomerular segregation is regulated by the adhesive/repulsive axonal interactions mediated by axon-sorting molecules whose expression levels are determined by expressed OR molecules7. The axon-sorting molecules involved in glomerular segregation are expressed in a position-independent mo...

Watch this Scientific Journal Video about Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes at JoVE.com

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes (Video) | JoVE

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Research

JoVE Journal

Neuroscience

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording (Video) | JoVE

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

Isolating Nasal Olfactory Stem Cells from Rodents or Humans (Video) | JoVE

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo (Video) | JoVE

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines (Video) | JoVE

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons (Video) | JoVE

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals (Video) | JoVE

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans (Video) | JoVE

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna (Video) | JoVE

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

Olfactory Assays for Mouse Models of Neurodegenerative Disease

Olfactory Assays for Mouse Models of Neurodegenerative Disease (Video) | JoVE

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb (Video) | JoVE

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...