Name: rapid in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed brain of rats with serotonin syndrome
Uploaded: 2015-09-23T15:00:00
Description: Watch this Scientific Journal Video about Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome at JoVE.com

इस अध्ययन एनआईएच अनुदान (R15DA029863) द्वारा समर्थित किया गया था, फ्लोरिडा अटलांटिक विश्वविद्यालय स्नातक अनुसंधान अनुदान (M30014) और पशु चिकित्सा अनुसंधान अनुदान के रॉस विश्वविद्यालय के स्कूल। हम इस काम के लिए (±) 3,4 methylenedioxymethamphetamine (± एमडीएमए) प्रदान करने के लिए नशीली दवाओं के सेवन पर राष्ट्रीय संस्थान (रॉकविल, एमडी) का शुक्रिया अदा करना चाहते हैं।

नीचे वर्णित पशु उपयोग प्रक्रियाओं पशु चिकित्सा के रॉस विश्वविद्यालय के स्कूल में संस्थागत पशु की देखभाल और उपयोग समिति (IACUC) और फ्लोरिडा अटलांटिक विश्वविद्यालय द्वारा अनुमोदित किया गया। बाँझ तकनीक औ?...

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One of the major concerns of in situ hybridization test is to choose appropriate techniques used for RNA preservation because of RNase enzymes. It is well known that these enzymes are widely present in the cells and environment which can affect the results. However, enzyme activity can be quickly distinguished by placing the tissue in the dry ice/alcohol solution5,12. Although quick preservation is critical for hybridization using a riboprobe13, little is known about experimental conditions...

Serotonin (5-hydroxytryptamine; 5-HT) syndrome is an acute neurologic disorder caused by 5-HT-promoting drugs such as antidepressants1, while also occurring in situations of MDMA use for recreational purposes2. Molecular mechanisms responsible for mood swings, learning and memory deficits that occur in association with the acute syndrome are not well understood3,4. In situ hybridization is a powerful research tool allowing the detection and quantification of specific mRNAs expressed potentially at a single-cell level. The conventional way to perform in situ hybridization is to utilize a radioactive-labeled riboprobe that specifically hybridizes the gene of interest. However, a major drawback is that the method requires complicated and time-consuming probe preparation and hybridization steps, as well as access to fresh frozen tissues maintained in an RNase-free environment5,6. 
Oligonucleotide probes have been recently developed to hybridize shorter RNA fragments than those required for riboprobes7. Furthermore, the probes produce a low background signal without sacrificing specificity8. This newly-developed probe technology can be used in situ hybridization on paraformaldehyde-prefixed brain tissues commonly available in immunocytochemical laboratories.
Here, we describe a protocol for in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed rat brain and compare findings with those noted in a fresh frozen brain5,6. This protocol is used to test the hypothesis that MDMA substance abuse causes changes in 5-HT2A receptor gene htr2a mRNA in the brain. We began the procedure with MDMA treatment followed by paraformaldehyde tissue perfusion of the animal, in situ hybridization of the thr2a probe, and data analysis. Note that dapB (the bacterial gene coding for dihydrodipicolinate reductase) is used as a negative control, and ppiB (housekeeping gene coding for peptidylprolyl isomerase B) as a positive control.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr>
			<td>RNAscope Negative Control Probe-DapB</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310098</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Pretreat 4</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>320046</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope 2.0 HD Reagent Kit - Red</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310036</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Probe - Rn-Ppib</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>313921</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Htr2a</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>300031</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM 1850</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>VWR</td>
			<td>88032-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Thermo Fisher Scientific</td>
			<td>222000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus microscope slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic pen</td>
			<td>Vector</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>Provis AX70</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Oligonucleotide आरएनए जांच (&lt;25 NT) का उपयोग करना, संकरण paraformaldehyde-उपसर्ग और ताजा जमे हुए दिमाग से तैयार हाईपोथेलेमिक कोशिकाओं में लाल डॉट्स के रूप में पता लगाया जा सकता है। htr2a mRNA के अणुओं (ठोस तीर द्वारा संकेत) कुछ कोश...

Watch this Scientific Journal Video about Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome at JoVE.com

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.

तीव्र<em&gt; बगल में</emसेरोटोनिन सिंड्रोम के साथ चूहे की Paraformaldehyde-उपसर्ग मस्तिष्क पर oligonucleotide जांच का उपयोग कर&gt; संकरण

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

Research

JoVE Journal

Neuroscience

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

डबल प्रतिदीप्ति बगल में</emताजा ब्रेन धारा में> संकरण

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

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

टीएमएस: थीटा - फट प्रोटोकॉल का उपयोग करने के लिए नाजुक एक्स सिंड्रोम और Autism के साथ व्यक्तियों में plasticity की Mechasnism अन्वेषण

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

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

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

Lipophilic डाई संयोजन, बगल में संकरण, immunohistochemistry, और प्रोटोकॉल

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

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

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

इसके बाद wholemount के लिए पूरी स्पाइनल कॉर्ड चोट और मस्तिष्क विच्छेदन प्रोटोकॉल<em&gt; बगल में</em&gt; लारवल सागर एक प्रकार की मछली में संकरण

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

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

उच्च संकल्प का उपयोग मस्तिष्क वर्गों में Axonally स्थानीयकृत mRNAs का पता लगाने<em&gt; बगल में</em&gt; संकरण

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

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

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

डबल प्रतिदीप्ति का मेल<em&gt; बगल में</em&gt; माउस मस्तिष्क वर्गों में तीन जीनों की अभिव्यक्ति की जांच के लिए Immunolabelling साथ संकरण

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

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

एक उपन्यास पद्धति का उपयोग करते हुए मुक्त नवजात चूहों को उड़ाने में ईईजी रिकॉर्डिंग

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

तीव्र मस्तिष्क स्लाइस में एटीपी या सेरोटोनिन की ओर Microglial प्रक्रियाओं ' आकर्षण के दो-फोटॉन इमेजिंग

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

हल्के अभिघातजन्य मस्तिष्क की चोट के साथ चूहों के हिप्पोकैम्पस में उन्नत प्रसार इमेजिंग

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

घूर्णन त्वरण के आधार पर चूहों में डिफ्यूज एक्सोनल ब्रेन इंजरी का प्रेरण

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...