This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23700412, 25122707 and 26670090 to S.Y.).

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at Hamamatsu University School of Medicine.
1. Preparation of Matrices
<ol>
	<li>Boil 4-8 silicone matrices in a microwave or on a hot plate for 5 min and allow them to dry completely for 1 h under laminar flow (striped side up). 
	Note: The following procedures should be performed under laminar flow.</li>
	<li>Blow compressed air or use transparent sticky tape to remove any dust from the striped side of the matrices. Keep the striped side clean so it can firmly adhere to the plate surface.</li>
	<li>Carefully place the matrices onto 6-cm plastic dishes by pressing with a finger (one matrix per dish; stripe side down). Avoid getting air bubbles between the matrix and the dish. Mark the location of the stripes on the bottom side of the culture dish.</li>
	<li>If the matrix fails to attach, repeat from step 1.2.</li>
</ol>
2. Stripe Generation and Laminin Coating for Neuron Culture
<ol>
	<li>Prepare fluorescently labeled recombinant protein (25 &#181;l each for injection [step 2.2] or 100 &#181;l each for the placement method [step 2.2.1]) by mixing the Fc-tagged protein (10-50 &#181;g/ml) and a fluorescent dye-conjugated anti-human Fc antibody (1 to 3 ratio: 30-150 &#181;g/ml) in phosphate buffered saline (PBS). Then, incubate the mixture for 30 min at RT.</li>
	<li>Using a 22-G syringe, inject 25 &#181;l of the recombinant protein prepared in the previous step (2.1) through the small hole on the side of the matrix (arrows in Figure 1A, 1C, and 1E).
	<ol>
		<li>Alternatively, place 100 &#181;l of the recombinant protein into the top slit of the matrix and aspirate approximately half of the solution from the small hole (arrows in Figure 1A, 1C, and 1E), so that the recombinant protein remains in the striped regions.</li>
	</ol>
	</li>
	<li>Incubate the dish for 30 min at 37 &#176;C in a cell culture incubator.</li>
	<li>Place 300 &#181;l of PBS into the top slit (Figure 1B) and aspirate from the small hole located on the side of the matrix (arrows in Figure 1A, C and E) to remove unattached recombinant proteins. Do not aspirate the PBS completely, because the proteins will dry. Repeat this step twice. (3 times total)</li>
	<li>Carefully remove the matrix and immediately place ~100 &#181;l of control protein (10-50 &#181;g/ml; Fc) to cover the entire striped area, creating an alternate coating. Then, incubate the dish for 30 min at 37 &#176;C in the cell culture incubator.</li>
	<li>After washing the dish surface three times with PBS, coat it with ~100 &#181;l of 20 &#181;g/ml laminin in PBS. 
	Note: Laminin should be thawed on ice to prevent solidification.</li>
	<li>Incubate the dish for 1 h at 37 &#176;C in the cell culture incubator.</li>
	<li>Wash the dish surface three times with PBS and add culture medium. Place the coated dish with culture medium in a 5% CO2 incubator at 37 &#176;C until use.</li>
</ol>
3. Culturing Hippocampal Neurons from E15.5 Mouse Embryos
<ol>
	<li>Euthanize mother via cervical dislocation and isolate three E15.5 embryos.</li>
	<li>Cut the skin and skull sagittally at the midline of the head with scissors, and remove the brain using a small spoon. Transfer the brain carefully to a 60-mm Petri dish containing 3 ml of Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Using a scalpel, cut out the hemispheres including cortex, hippocampus and striatum (Figure 2A). Then, dissect out the hippocampal region by carefully removing the meninges, and transfer the dissected hippocampal tissues to a 15-ml centrifuge tube containing 2 ml of HBSS solution, on ice (Figure 2B-C). Collect 6 hippocampi from 3 embryos in the tube, which are sufficient for culturing neurons in 8 dishes.</li>
	<li>Replace the HBSS solution with trypsin/EDTA solution (2 ml) and incubate the tube at 37 &#176;C for 15 min in a water bath. 
	Note: Aspirate HBSS without centrifugation and do not decant the HBSS solution by tilting the tube.</li>
	<li>Neutralize the trypsin activity by adding 500 &#181;l of fetal bovine serum (FBS).</li>
	<li>Centrifuge the mixture at 100 x g for 5 min. Remove the supernatant and wash the pellet with 2 ml of culture medium. Repeat this step once more.</li>
	<li>Pipet the hippocampi up and down 10 times in culture medium using a 1,000-&#181;l tip with a large hole (cut-tip) to dissociate the tissue.</li>
	<li>Change to a regular (uncut) 1,000-&#181;l tip and pipet the dissociated tissue up and down to obtain a single-cell suspension.</li>
	<li>Separate the single cells by passing them through an 80-100-&#181;m mesh cell strainer.</li>
	<li>Centrifuge the filtered single cell suspension at 150 x g for 5 min.</li>
	<li>Remove the supernatant by aspirating, and resuspend the pellet (neurons) in 2 ml of culture medium.</li>
	<li>Count the cells using a hemocytometer with trypan blue staining to determine the density and viability. Then, plate 10,000 neurons in 150 &#181;l of culture medium for each set of stripes.The suspension should be carefully placed to cover the entire stripe region.</li>
</ol>
4. Visualization of Cell Bodies and Axons by Immunofluorescence Staining using an Anti-Tau1 Antibody
<ol>
	<li>After 24 h of culture, aspirate the medium without disturbing the adherent cells, and fix the neurons with pre-warmed 4% paraformaldehyde (PFA)/4% sucrose/PBS at RT&#160;for 15 min.</li>
	<li>Wash the plate surface 3 times with PBS. If desired, use a water-repellant pen to draw a circle around the striped region to decrease the amount of antibody needed.</li>
	<li>Permeabilize the neurons with 0.3% Triton X-100/PBS for 5 min.</li>
	<li>Incubate the neurons with blocking solution containing 3% donkey serum and 0.1% Triton X-100 for 30 min followed by incubation with an anti-Tau1 antibody (1:200) in 1% donkey serum/0.1% Triton X-100/PBS O/N at 4 &#176;C.</li>
	<li>Wash the plate surface 3 times with PBS. Incubate the neurons with a fluorophore-conjugated anti-mouse IgG antibody in 1% bovine serum albumin (BSA)/0.1% Triton X-100/PBS for 30 min.</li>
	<li>Wash the plate surface 3 times with PBS. Cover the striped region with an 18 mm x 18 mm cover glass using 50 &#181;l of antifade solution. avoid air bubbles, which interfere with the imaging.</li>
	<li>Obtain fluorescent images with a fluorescence microscope using the appropriate system, e.g., x10 objective lens, filter sets for GFP and RFP, and 1,000-msec exposure time (Figure 3).</li>
</ol>

<ol>
	<li>Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+axon+guidance.">Molecular mechanisms of axon guidance.</a> Science. 298 (5600), 1959-1964 (2002).</li><li>Bashaw, G. J., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+from+axon+guidance+receptors.">Signaling from axon guidance receptors.</a> Cold Spring Harb Perspect Biol. 2 (5), 001941 (2010).</li><li>Hong, K., Nishiyama, M., Henley, J., Tessier-Lavigne, M., Poo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling+in+the+guidance+of+nerve+growth+by+netrin-1.">Calcium signalling in the guidance of nerve growth by netrin-1.</a> Nature. 403 (6765), 93-98 (2000).</li><li>Ming, G. -. l., Henley, J., Tessier-Lavigne, M., Song, H. -. j., Poo, M. -. m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+Activity+Modulates+Growth+Cone+Guidance+by+Diffusible+Factors.">Electrical Activity Modulates Growth Cone Guidance by Diffusible Factors.</a> Neuron. 29 (2), 441-452 (2001).</li><li>Dudanova, I., 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=Genetic+evidence+for+a+contribution+of+EphA:ephrinA+reverse+signaling+to+motor+axon+guidance.">Genetic evidence for a contribution of EphA:ephrinA reverse signaling to motor axon guidance.</a> J Neurosci. 32 (15), 5209-5215 (2012).</li><li>Ye, B. Q., Geng, Z. H., Ma, L., Geng, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slit2+regulates+attractive+eosinophil+and+repulsive+neutrophil+chemotaxis+through+differential+srGAP1+expression+during+lung+inflammation.">Slit2 regulates attractive eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 expression during lung inflammation.</a> J Immunol. 185 (10), 6294-6305 (2010).</li><li>Ly, 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=DSCAM+is+a+netrin+receptor+that+collaborates+with+DCC+in+mediating+turning+responses+to+netrin-1.">DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1.</a> Cell. 133 (7), 1241-1254 (2008).</li><li>Hata, K., Kaibuchi, K., Inagaki, S., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unc5B+associates+with+LARG+to+mediate+the+action+of+repulsive+guidance+molecule.">Unc5B associates with LARG to mediate the action of repulsive guidance molecule.</a> J Cell Biol. 184 (5), 737-750 (2009).</li><li>Egea, J., 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=Regulation+of+EphA+4+kinase+activity+is+required+for+a+subset+of+axon+guidance+decisions+suggesting+a+key+role+for+receptor+clustering+in+Eph+function.">Regulation of EphA 4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function.</a> Neuron. 47 (4), 515-528 (2005).</li><li>von Philipsborn, A. C., Lang, S., Jiang, Z., Bonhoeffer, F., Bastmeyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-Bound+Protein+Gradients+for+Cell+Culture+Fabricated+by+Microfluidic+Networks+and+Microcontact+Printing.">Substrate-Bound Protein Gradients for Cell Culture Fabricated by Microfluidic Networks and Microcontact Printing.</a> Sci Signal. , (2007).</li><li>Jackman, R., Wilbur, J., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+submicrometer+features+on+curved+substrates+by+microcontact+printing.">Fabrication of submicrometer features on curved substrates by microcontact printing.</a> Science. 269 (5224), 664-666 (1995).</li><li>Mrksich, M., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+self-assembled+monolayers+to+understand+the+interactions+of+man-made+surfaces+with+proteins+and+cells.">Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells.</a> Annu Rev Biophys Biomol Struct. 25, 55-78 (1996).</li><li>Walter, J., Kern-Veits, B., Huf, J., Stolze, B., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+position-specific+properties+of+tectai+cell+membranes+by+retinal+axons+in+vitro.">Recognition of position-specific properties of tectai cell membranes by retinal axons in vitro.</a> Development. 101, 685-696 (1987).</li><li>Knoll, B., Weinl, C., Nordheim, A., Bonhoeffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripe+assay+to+examine+axonal+guidance+and+cell+migration.">Stripe assay to examine axonal guidance and cell migration.</a> Nat Protoc. 2 (5), 1216-1224 (2007).</li><li>Weschenfelder, M., Weth, F., Knoll, B., Bastmeyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stripe+assay:+studying+growth+preference+and+axon+guidance+on+binary+choice+substrates+in+vitro.">The stripe assay: studying growth preference and axon guidance on binary choice substrates in vitro.</a> Methods Mol Biol. 1018, 229-246 (2013).</li><li>Seiradake, E., 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=Structure+and+functional+relevance+of+the+Slit2+homodimerization+domain.">Structure and functional relevance of the Slit2 homodimerization domain.</a> EMBO Rep. 10 (7), 736-741 (2009).</li><li>Gebhardt, C., Bastmeyer, M., Weth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+of+ephrin/Eph+forward+and+reverse+signaling+as+the+driving+force+of+adaptive+topographic+mapping.">Balancing of ephrin/Eph forward and reverse signaling as the driving force of adaptive topographic mapping.</a> Development. 139 (2), 335-345 (2012).</li><li>Atapattu, L., 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=Antibodies+binding+the+ADAM10+substrate+recognition+domain+inhibit+Eph+function.">Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.</a> J Cell Sci. 125, 6084-6093 (2012).</li><li>Stark, D. A., Karvas, R. M., Siegel, A. L., Cornelison, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eph/ephrin+interactions+modulate+muscle+satellite+cell+motility+and+patterning.">Eph/ephrin interactions modulate muscle satellite cell motility and patterning.</a> Development. 138 (24), 5279-5289 (2011).</li><li>Seiradake, E., 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=FLRT+Structure:+Balancing+Repulsion+and+Cell+Adhesion+in+Cortical+and+Vascular+Development.">FLRT Structure: Balancing Repulsion and Cell Adhesion in Cortical and Vascular Development.</a> Neuron. 84 (2), 370-385 (2014).</li><li>Yamagishi, 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=FLRT2+and+FLRT3+act+as+repulsive+guidance+cues+for+Unc5-positive+neurons.">FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons.</a> EMBO J. 30 (14), 2920-2933 (2011).</li></ol>

The authors have nothing to disclose.

This protocol describes a stripe assay that uses recombinant protein and dissociated neurons from the E15.5 mouse hippocampus. This assay allows the efficient observation of repulsive, attractive, or neutral responses of neurons to a recombinant protein of interest placed in a striped pattern. A major advantage of this protocol is the simple method for generating the stripes, in which the protein is directly printed onto the surface of a plastic dish, compared to the traditional method, which requires special matrices, a...

Axon guidance is the process by which newly formed neurons send axons to their target during development of the nervous system1,2. Developing axons carry a highly motile structure at their tip called the growth cone. The growth cone senses extracellular cues to navigate the axon&#39;s path. Guidance molecules, such as slit, semaphorin, and ephrins, can attract or repel axons depending on their interaction with suitable receptors and co-receptors on the axon1,3,4. The activated receptors transfer signals to the growth cone that affect its cytoskeletal organization for axon and growth-cone movements.
Various methods have been developed to evaluate the action of attractant and repellent molecules. Chemo-attractants and repellents can be administered into the growth/culture medium with a gradient concentration (e.g., Dunn&#39;s chamber or &#181;-slides)5,6, in a highly concentrated spot by micro-pipette (e.g., turning assay)7 or at a homogenous concentration by bath application (e.g., growth cone collapse assay)8,9.
Other methods include a stripe assay or microcontact printing (&#181;CP), in which a chemo-attractant or repellent is coated on the surface of a plate as a substrate10-12. Thestripe assay was originally developed by Bonhoeffer and colleagues in 1987 to analyze topographical mapping in the chick retino-tectal system13. The original method required a vacuum system to coat proteins onto polycarbonate nucleopore membranes using striped and meshed matrices. In later versions, the recombinant proteins were directly printed on the surface of a culture plate in a striped pattern using narrow slit silicon matrices14,15. Recently, various research groups have successfully applied this stripe assay to the analysis of axon guidance molecule activities16-21.
Here, we present the detailed protocol for a stripe assay that measures the attraction or repulsion of axon guidance molecules for dissociated hippocampal neurons. Notably, this method can be applied in minimally equipped laboratory settings. For this assay, alternating stripes of a fluorescently labeled substrate and a control protein are generated on a plastic dish using a silicon matrix with 90-&#181;m slits and coated with laminin. In our demonstration, dissociated hippocampal neurons from E15.5 mice were cultured on alternating stripes of recombinant ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control Fc protein21. After 24 h of culture, both the axons and cell bodies of the neurons were strongly repelled from the FLRT2 stripes. Staining with an anti-Tau1 antibody revealed that ~90% of the neurons were distributed on the Fc-coated regions, compared to ~10% on the FLRT2-Fc, indicating that FLRT2 has a strong repulsive function for hippocampal neurons21.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Violamo&#160;</td>
			<td>1-3500-01</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde (PFA)</td>
			<td>Nacalai</td>
			<td>01954-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat anti-human IgG antibody</td>
			<td>Thermo Scientific</td>
			<td>A11013</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-mouse IgG antibody</td>
			<td>Thermo Scientific</td>
			<td>A-21203</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Anti-Tau1 antibody</td>
			<td>Chemicon</td>
			<td>MAB3420</td>
			<td>Dilution 1/200</td>
		</tr>
		<tr>
			<td>Antifade</td>
			<td>Thermo Scientific</td>
			<td>P7481</td>
			<td>Alternative mounting media may be used</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Scientific</td>
			<td>17504-044</td>
			<td>Dilution 1/50</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>01-2030-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 um</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugation machine</td>
			<td>Kubota</td>
			<td>2410</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass 18mmx18mm</td>
			<td>Matsunami</td>
			<td>18x18 mm No. 1</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO pen</td>
			<td>DAKO</td>
			<td>S2002</td>
			<td>Alternative water-repellent pen may be used</td>
		</tr>
		<tr>
			<td>Disposable scalpel</td>
			<td>Feather</td>
			<td>2975#11</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorecent microscope</td>
			<td>Nikon</td>
			<td>E600</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps No. 5</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Scientific</td>
			<td>35050-061</td>
			<td>Dilution 1/200</td>
		</tr>
		<tr>
			<td>Hamilton Syringe</td>
			<td>Hamilton</td>
			<td>805N</td>
			<td>22 gauge, 50 uL</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Scientific</td>
			<td>14170-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG, Fc Fragment</td>
			<td>Jackson</td>
			<td>009-000-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Scientific</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Thermo Scientific</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Nacalai</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15070-063</td>
			<td>Dilution 1/100</td>
		</tr>
		<tr>
			<td>Plastic culture dish, 60 mm</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Matrices</td>
			<td></td>
			<td></td>
			<td>Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu)</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Tip, 1000 uL</td>
			<td>Watson</td>
			<td>125-1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent sticky tape</td>
			<td>Tesa</td>
			<td>57315</td>
			<td>Alternative sticky tape may be used</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4%</td>
			<td>Bio-Rad</td>
			<td>145-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Thermo Scientific</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture medium</td>
			<td></td>
			<td></td>
			<td>Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin.</td>
		</tr>
	</tbody>
</table>

stripe assay to study the attractive or repulsive activity of a protein substrate using dissociated hippocampal neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Dissociated hippocampal neurons from E15.5 mice were plated and cultured for 24 h on stripes of fluorescently labeled control Fc (Figure 3A-C) or FLRT2-Fc (Figure 3D-F) alternating with non-labeled control Fc. In both cases, the neurons were aggregated and extended their axons as bundles. On the control Fc/Fc stripes, the neurons were distributed evenly on the fluorescently labeled and non-labeled stripes, and they extended their axons in random direction...

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Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

stripe-assay-to-study-attractive-or-repulsive-activity-protein

Research

JoVE Journal

Neuroscience

10.5K Views.  Hamamatsu University School of Medicine. Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons. 

Video: Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...