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

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.

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

The overall goal of this immunohistochemical staining method is to visualize combined expression of axon sorting molecules at the axon termini of olfactory sensory neurons. This method can help characterize key processes in the field of olfactory development, such as axon guidance, synapse formations, and the regeneration of olfactory sensory neurons. The main advantage of this technique is that it allows scientists to compare and analyze the expression patterns of axon sorting molecules in olfactory bulb without sustaining variation between sections.Visual demonstration of this method is critical as the embedding step is difficult to learn, because the position and angle of the olfactory tissues sample in the mold are hard to describe in words. Start with heads from mice of one to two weeks of age that have been fixed by intracardiac perfusion of 4%paraformaldehyde. Insert a scissor blade into the space between the upper and lower teeth, and cut horizontally to remove the lower jawbone.Then trim away excess tissue with forceps and scissors, to leave the olfactory tissue, including the olfactory bulb and the olfactory epithelium. Immerse the olfactory tissue in PBS to remove air in the nasal cavity, then ake a vertical cut to remove the posterior part of the brain, and place the olfactory tissue on the bottom of an embedding mold with the cut end face down. Fill the mold with optimal cutting temperature compound, and then immerse in liquid nitrogen.After the tissue and OCT is completely frozen, equilibrate the tissue in the cryostat at 20 degrees celsius for one hour. Now make serial parasaggital sections of the olfactory bulb with the cryostat, and collect them by sticking to MAS coated glass slides. After sticking, dry the slides immediately with a blow dryer.Begin immunostaining by washing the dried slides with PBS for five minutes at room temperature. Block nonspecific binding sites by incubating the slides for one hour with 5%blocking solution at room temperature. Then add 400 microliters of a cocktail of primary antibodies diluted in 1%blocking solution to each slide.Incubate the slides overnight at room temperature. The next day, discard the primary antibody solution and wash the slides with PBST three times for five minutes each at room temperature. Then add 400 microliters of a cocktail of secondary antibodies and PBS to each slide.Protect from light and incubate for one hour at room temperature. Following the incubation, discard the solution and again wash the slides three times for five minutes each with PBS at room temperature. Finally, mount cover slips on the slides by applying two drops of mounting medium to each slide, placing the cover slip, and removing air bubbles.Obtain fluorescent images with the fluorescence microscope using the appropriate filter for each fluorophor. Adjust the exposure time to obtain fluorescent signals of the image without saturation. Define the glomerular structure by immunofluorescent signals of GLUT2.Use image J to measure staining intensities of axon sorting molecules within glomeruli. This image shows a parasaggital olfactory bulb section from a two week old mouse immunostained with antibodies against VGLUT2, Kirrel2 shown in red, Semaphorin 7A shown in green, and OLPC in blue. The merged image demonstrates that the axon sorting molecules involved in glomerular segregation are expressed in a position-independent mosaic pattern.Each glomerulus is defined by fluorescent signals of the presynaptic marker VGLUT2. The glomerular structures are surrounded by dashed lines. Signal intensities of axon-sorting molecules were measured in each glomerulus.As seen here, axon sorting molecules are differentially expressed in each glomerulus. For example, glomerulus 3 has high levels of OLPC, intermediate levels of Semaphorin 7A, and lower levels of Kirrel2, and this difference can be quantified by measuring the staining intensity. Whereas glomerulus 4 has high levels of OLPC, and lower levels of Kirrel2 and Semaphorin 7A.Again, this difference can be quantified by measuring the staining intensity. While attempting this procedure, it's important to remember to omit post-fixation with PFA, and treatment with sucrose solution, which are normally recommended. After watching this video, you should have a good understanding of how to place more glomeruli on the single olfactory bulb section, and how to perform high quality immunostaining with multiple antibodies, which can be applied to other brain areas.Don't forget to hope, this method will help you succeed in your future experiments.

quadruple-immunostaining-olfactory-bulb-for-visualization-olfactory

Research

JoVE Journal

Neuroscience

7.0K Aufrufe.  The University of Tokyo. The overall goal of this immunohistochemical staining method is to visualize combined expression of axon sorting molecules at the axon termini of olfactory sensory neurons. This method can help characterize key processes in the field of olfactory development, such as axon guidance, synapse formations, and the regeneration of olfactory sensory neurons. The main advantage of this technique is that it allows scientists to compare and analyze the expression patterns of axon sorting molecules in o...