Confocal imágenes se obtuvieron en la Universidad de Iowa Carver Centro de Imagen. El financiamiento fue proporcionado por una beca SBIR (MH079805) para apoyar BF adicionales para la cría de los ratones de este proyecto fue proporcionado por NIDCD (5R01DC00559007) para BF

<ol>
	<li>Jensen-Smith, H., Gray, B., Muirhead, K., Ohlsson-Wilhelm, B., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-distance+three-color+neuronal+tracing+in+fixed+tissue+using+NeuroVue+dyes.">Long-distance three-color neuronal tracing in fixed tissue using NeuroVue dyes.</a> Immunol Invest. 36, 763-789 (2007).</li><li>Tonniges, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-+and+bio-physical+characterization+of+novel+violet+and+near-infrared+lipophilic+fluorophores+for+neuronal+tracing.">Photo- and bio-physical characterization of novel violet and near-infrared lipophilic fluorophores for neuronal tracing.</a> Journal of Microscopy. , (2010).</li><li>Bartheld, C. S. v. o. n., Cunningham, D. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+tracing+with+DiI:+decalcification,+cryosectioning,+and+photoconversion+for+light+and+electron+microscopic+analysis.">Neuronal tracing with DiI: decalcification, cryosectioning, and photoconversion for light and electron microscopic analysis.</a> J Histochem Cytochem. 38, 725-733 (1990).</li><li>Gurung, B., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+embryonic+midbrain+thalamic+and+thalamic+auditory+connection+development+in+mice+as+revealed+by+carbocyannine+dye+injection.">Time course of embryonic midbrain thalamic and thalamic auditory connection development in mice as revealed by carbocyannine dye injection.</a> J Comp Neurol. 479, 309-327 (2004).</li><li>Fritzsch, B., Nichols, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiI+reveals+a+prenatal+arrival+of+efferents+at+the+differentiating+otocyst+of+mice.">DiI reveals a prenatal arrival of efferents at the differentiating otocyst of mice.</a> Hear Res. 65, 51-60 (1993).</li><li>Jahan, I., Kersigo, J., Pan, N., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurod1+suppresses+hair+cell+differentiation+in+ear+ganglia+and+regulates+hair+cell+subtype+development+in+the+cochlea.">Neurod1 suppresses hair cell differentiation in ear ganglia and regulates hair cell subtype development in the cochlea.</a> PloS ONE. 5, e11661-e11661 (2010).</li></ol>

Tejido de ratón fue recopilada de conformidad con las directrices y normas establecidas por la Universidad de Iowa Cuidado de Animales institucional y el empleo (ACURF 0.804.066).

En este vídeo, se demuestra un método para combinar cuatro técnicas para maximizar la recogida de datos y la cohesión mediante la correlación de los datos dentro de un determinado animal (figura 3). Este enfoque reducirá la cantidad de cría y por lo tanto el tiempo necesario para obtener los datos publicables y mejorar la información sobre la co-localización y los efectos correlativos de los mutantes. A pesar de embriones de ratones se utilizaron en este vídeo, ratón adulto, así como otros animales como el pollo, Xenopus y pez cebra puede ser analizada utilizando estas técnicas también. Cuando se utilizan otras especies digestión de proteinasa k puede ser necesario modificar. Aquí usamos diferentes tintes fluorescentes NeuroVue para etiquetar específicamente hasta tres poblaciones neuronales de interés. La ventaja de estos tintes es que pueden ser utilizados en los ratones mutantes, que puede ser problemático cuando se basa únicamente en técnicas de inmunohistoquímica que pueden o no pueden ser afectados por las mutaciones a analizar, y que ellos llaman neuronas retrógradamente y anterogradely 5. Un estudio reciente también ha aumentado el número de poblaciones de neuronas que pueden ser etiquetados de forma simultánea a seis 2. Después, el colorante lipofílico seguimiento del mismo tejido de interés se pueden analizar utilizando hibridación in situ, para el análisis de la transcripción de genes y pueden ser posteriormente analizados mediante técnicas de inmunohistoquímica para la distribución de las proteínas. Este último análisis se puede complementar con la histología detallada que se puede añadir a la caracterización fenotípica 6. Este análisis multifactorial tiene el potencial para obtener nuevos conocimientos a través del análisis correlativo en el mismo animal que de lo contrario puede ser improbable, o al menos en la necesidad de protocolos más extensa y la cría de ratón.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>in situ Hybridization:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PC DIG wash + block buffer set</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1585762</td>
<td>&nbsp;</td>
<tr>
<td>Premix 20x SSC Buffer</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1666681</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K 20mg/ml</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM2546</td>
<td>&nbsp;</td>
<tr>
<td>Diethyl Pyrocorbonate (DEPC) </td>
<td>&nbsp;</td>
<td>RPI</td>
<td>D43060</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9037</td>
<td>&nbsp;</td>
<tr>
<td>Dextran Sulfate</td>
<td>&nbsp;</td>
<td>RPI</td>
<td>D20020</td>
<td>&nbsp;</td>
<tr>
<td>BM Purple </td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11442074001</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Dig-AP Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11093274910</td>
<td>&nbsp;</td>
<tr>
<td>RNase A enzyme 10mg/ml</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>EN0531</td>
<td>&nbsp;</td>
<tr>
<td>RNase Away</td>
<td>&nbsp;</td>
<td>RPI</td>
<td>7003</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA 10mg/ml</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15632-011</td>
<td>&nbsp;</td>
<tr>
<td>Stericup Filter Unit 0.22&mu;m; 500ml</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SCGVU05RE</td>
<td>&nbsp;</td>
<tr>
<td>Filter Discs 0.2&mu;m; 25mm</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>6780-2502</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline PH 7.4 packets</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-5368</td>
<td>&nbsp;</td>
<tr>
<td>Immunohistochemistry:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Goat Serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G9023</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8532</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst Dye</td>
<td>&nbsp;</td>
<td>Polysciences Inc.</td>
<td>9460</td>
<td>&nbsp;</td>
<tr>
<td>Histology:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Poly/Bed 812 Embedding Media/DMP-30 Kit </td>
<td>&nbsp;</td>
<td>Polysciences Inc. </td>
<td>08792-1</td>
<td>&nbsp;</td>
<tr>
<td>Propylene oxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>110205</td>
<td>&nbsp;</td>
<tr>
<td>Gluteraldehyde Solution 50%</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G151</td>
<td>&nbsp;</td>
<tr>
<td>DPX Mountant</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>44581</td>
<td>&nbsp;</td>
<tr>
<td>Rocker</td>
<td>&nbsp;</td>
<td>MidSci</td>
<td>55D1114</td>
<td>&nbsp;</td>
<tr>
<td>Heat Block</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-715-125D</td>
<td>&nbsp;</td>
<tr>
<td>Rotator</td>
<td>&nbsp;</td>
<td>Thermo</td>
<td>400110Q</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set to 60&deg;</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-690-625D</td>
<td>&nbsp;</td>
<tr>
<td>Dye tracing:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscissors</td>
<td>&nbsp;</td>
<td>Geuder</td>
<td>G-19775</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps</td>
<td>&nbsp;</td>
<td>E.M.S</td>
<td>72680-F</td>
<td>&nbsp;</td>
<tr>
<td>NeuroVue dye: Maroon, Red, Jade</td>
<td>&nbsp;</td>
<td>MTTI</td>
<td>FS-100(1,2,6)</td>
<td>&nbsp;</td>
<tr>
<td>1X phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-5368</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting scope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>M205 FA</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set to 36&deg;</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-690-506DQ</td>
<td>&nbsp;</td>
<tr>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>LSM 510</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>Surgipath</td>
<td>00240</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>&nbsp;</td>
<td>Surgipath</td>
<td>00106</td>
<td>&nbsp;</td>
<tr>
<td>RPI</td>
<td>&nbsp;</td>
<td>Glycerol</td>
<td>G22025-0.5</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (4% and 0.4%)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>T353-500</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

combining lipophilic dye, in situ hybridization, immunohistochemistry, and histology

Yendo más allá de la función de genes individuales para profundizarse en las redes reguladoras de genes requiere múltiples mutaciones combinadas en un solo animal. Este tipo de análisis de dos o más genes debe ser complementado con la hibridación in situ de otros genes, o inmunohistoquímica de las proteínas, tanto en el desarrollo de todo ello montado órganos o secciones de imagen de alta resolución de las alteraciones de la expresión celular y tisular. La combinación de múltiples alteraciones de genes requiere el uso de la creación o flipase eliminar condicionalmente genes y evitar la letalidad embrionaria. Sistemas de cría requiere mejorar dramáticamente el esfuerzo y el costo proporcional al número de genes mutados, con un resultado de muy pocos animales con el repertorio completo de las modificaciones genéticas deseadas. Amortización de la gran cantidad de esfuerzo y tiempo para obtener estos pocos ejemplares preciosos que son portadores de mutaciones múltiples requiere la optimización de los tejidos. Por otra parte, la investigación de un solo animal, con múltiples técnicas hace que sea más fácil de relacionar con los defectos genéticos borrado los perfiles de expresión. Hemos desarrollado una técnica para obtener un análisis más exhaustivo de un determinado animal, con la capacidad de analizar varias estructuras diferentes histológicamente reconocibles, así como la expresión de genes y proteínas todos los de la misma muestra en ambos órganos, y todo ello montado secciones. Aunque los ratones se han utilizado para demostrar la eficacia de esta técnica se puede aplicar a una amplia gama de animales. Para ello combinamos lipofílicas seguimiento medio de contraste, montar toda la hibridación in situ, inmunohistoquímica y la histología para extraer la máxima cantidad posible de datos.

Watch this Scientific Journal Video about Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology at JoVE.com

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Una combinación de diferentes técnicas para maximizar la recogida de datos a partir de tejido de ratón se presenta.

La combinación de colorantes lipofílicos, In situ</emHibridación>, inmunohistoquímica y la histología

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

combining-lipophilic-dye-situ-hybridization-immunohistochemistry

Research

JoVE Journal

Neuroscience

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

25.8K vistas.  University of Iowa. Una combinación de diferentes técnicas para maximizar la recogida de datos a partir de tejido de ratón se presenta.

Video: Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

La combinación del comportamiento de Endocrinología Experimental y Economía: Decisión de la testosterona y sociales que

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Investigación

Neurociencias

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Doble fluorescencia In situ La hibridación en las secciones del cerebro fresco

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Detección del genoma y transcripciones de un virus ADN persistente en los tejidos neuronales por Fluorescente In situ La hibridación Combinado con inmunotinción

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Lesiones de la Médula Espinal completo y Protocolo Disección del cerebro por Wholemount posterior<em&gt; In Situ</em&gt; La hibridación en larvas lamprea de mar

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

La detección de ARNm axón localizado en las secciones cerebrales Uso de alta resolución<em&gt; In Situ</em&gt; La hibridación

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

Rápido<em&gt; In Situ</em&gt; La hibridación usando sondas de oligonucleótidos en Cerebro paraformaldehído-prefijo de ratas con síndrome de serotonina

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

La combinación de doble fluorescencia<em&gt; In Situ</em&gt; La hibridación con Immunolabelling para la detección de la expresión de tres genes en las secciones de cerebro de ratón

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

La histología de masas para cuantificar la neurodegeneración en<em&gt; Drosophila</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Localización de Genes Receptor Odorante en Antenas Locustinas por RNA<em&gt; In Situ</em&gt; Hibridación

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Combinación de hibridación in situ de fluorescencia múltiple con inmunohistoquímica fluorescente en secciones de cerebro de ratón frescas, congeladas o fijas

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

La combinación de colorantes lipofílicos, In situ, inmunohistoquímica y la histología