This work was supported by the Alzheimer&#8217;s Association (NIRG-10-171721; to U.H.), National Institute of Mental Health (MH096702; to U.H.), National Institute of Neurological Disorders and Stroke (NS081333; to C.M.T.), and pilot study awards from the National Institute on Aging-funded Alzheimer&#8217;s Disease Research Center at Columbia University (AG008702; to J.B. and Y.Y.J.) that also supports the New York Brain Bank. We thank members of the Hengst laboratory for comments and discussions

<ol>
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The authors have nothing to disclose.

בדוח זה אנו מתארים את השימוש בטכנולוגית ISH ברזולוציה גבוהה בזיהוי של mRNA Atf4 המקומי axonally. מחקרים שפורסמו אלה וקודמים הראו כי טכנולוגיה זו היא תואמת עם זיהוי חלבון מבוסס נוגדנים ברקמות או אפילו עובר כל 33. חשוב לציין, זה כבר היה בשימוש לאחרונה לגילוי Arc mRNA ב...

גיוס mRNA axonal ותרגום מקומי לאפשר האקסונים להגיב לגירויים תאיים באופן 1 באופן זמני ומרחבית חריפה. סינתזת חלבון תוך-axonal היא הדרך הטובה ביותר להבין בהקשר של התפתחות המוח שבו משחק תפקידים מכריעים בצמיחה חרוט התנהגות 2-8, האקסון מפלס-הדרך 9-11 ומדרדר איתות 12,13. אבל הרבה פחות ידוע על המשמעות התפקודית של סינתזת חלבון axonal בתאי עצב פוסט-התפתחותית כאשר רמות ה- mRNA והריבוזום axonal מופחתות 14,15 מאוד. האקסונים חוליות בוגרים כבר מזמן חשבו להיות translationally פעיל 16. עם זאת, המחקרים אחרונים מצביעים על כך שהתרגום מקומי מחדש באקסונים בוגרים תחת מצבים פתולוגיים. לדוגמא, קבוצת משנה של mRNAs מגויס לאקסונים התחדשות הבאה פגיעה עצבית וסינתזה של חלבונים תוך-axonal נדרשים להתחדשות הנכונה של אקסונים אלה 17. בנוסף, חה הקבוצה שלנוים הוכיח כי mRNAs הספציפי מגויסים לאקסונים לאחר חשיפה מקומית לפפטיד מחלת אלצהיימר Aβ 1-42, ותרגום המקומי של ATF4 גורם השעתוק נדרשת כדי להפיץ את השפעות ניווניות של Aβ 1-42 מן האקסונים לסומה העצבית 18. לבסוף, ניתוחי תפוקה גבוהים גילו כי יש לי האקסונים בוגרים transcriptome יותר מורכבת ודינמית מהצפוי 18-21, במיוחד בתנאים פתולוגיים. לאור המחקרים הללו, שיטה רגישה וספציפית מאוד כדי לזהות mRNAs axonally מקומי במערכת העצבים הבוגרת יש צורך. חלק גדול מהעבודה על גיוס mRNA ותרגום מקומי באקסונים בוגרים שבוצע על נוירונים בתרבית. זה נכון במיוחד עבור transcriptome ניתוחים מאז שיטות culturing מיוחדות קיימות המאפשרות הבידוד של אקסונים מהתא הדנדריטי somato-18-20. למרות שמחקרים כאלה ניתנו תוספות יקרותight לתוך התפקיד של תרגום המקומי באקסונים בוגרים, השאלה אם נוירונים בתרבית מייצגים נאמנה את המצב בvivo או אם גיוס mRNA הוא תגובה אדפטיבית של אקסונים לתנאי culturing עדיין פתוח. מחקרים מעטים סיפקו ראיות לגיוס mRNA להבשיל האקסונים in vivo. לדוגמא, תמליל קידוד לחלבון סמן חוש הריח זוהה באקסונים בתאי עצב תחושתיים מבוגרים 22. Transgene המכיל &#39;UTR 3 של mRNA β-אקטין מועבר לאקסונים בתאי עצב במערכת עצבים היקפיים ומרכזיים בעכברים ומתורגם באופן מקומי לאחר תקופות התפתחותיות 23. B2 Lamin mRNA הוא מקומי לאקסונים רשתית בXenopues laevis ראשנים ודלדולה משפיע תחזוקת האקסון לאחר פיתוח axonal 21. הפרעה לתחבורה axonal של קידוד mRNA לIV מונואמין ציטוכרום C משנה עכבר התנהגות 24. לבסוף, Atf4 mRNA נמצא במבוגריםxons של עכברים ומוח אנושי בהקשר של Aβ 1-42 -induced ניוון מוחיים 18. ניתוחי transcriptome תפוקה גבוהה הוכיחו להיות שימושי כדי לזהות פרופילי mRNA באקסונים מבודדים במבחנה אבל יש מגבלות במחקרי vivo מאז בכל רקמות האקסונים לא נמצאים בבידוד, אבל התערבבו עם גופים עצביים תא, תאי גלייה וסוגי תאים אחרים. לפיכך, ניתוח כזה צריך להיות משולב עם טכניקות הדמיה שלאשר את לוקליזציה subcellular של mRNAs. RNA הכלאה באתר (RNA ISH) מאפשר זיהוי והדמיה של רצפי RNA ספציפיים בתאים וברקמות. עם זאת, מבחני RNA ISH המקוריים היו מתאימים רק לזיהוי RNAs הנרחב ביותר 25, שהוא לעתים רחוקות במקרה של mRNAs המקומי axonally. לעשורים האחרונים גוברים מאמצים כבר הכניס לתוך טכנולוגיות חדישות פיתוח המאפשרות זיהוי של mRNAs ברמת המולקולה בודדת <sup&gt; 25,26. לדוגמא, זינגר ועמיתיו פיתחו בדיקות ISH לזהות mRNAs בתאים בודדים, הכוללים 5 שאינם חופף שכותרתו fluorescently 50 מרס (לפרטים עיינו 27). ההבדל העיקרי בין השיטות שהוזכרו לעיל ואחד כאן תאר הוא שמאוחר יותר משתמש 20 Z-מבנה כפול (לא ליניארי) בדיקות, כי בדרך כלל היעד ~ אזור KB 1 של ה- RNA של הספציפיות הבטחת ריבית ורמות רקע נמוכות. אז בדיקות הם הכלאה עם רצפי מגבר קדם מגבר שסופו של דבר כותרת או מצומדות עם אנזימים המאפשרים תגובות chromogenic fluorescently. צעדי הגברה אלה משפרים את יחס אות לרעש בהשוואה לטכנולוגיות אחרות ISH 28. כאן אנו מתארים שני פרוטוקולים באמצעות RNAscope בשילוב גם עם immunocytochemistry הקרינה או עם צבעים היסטולוגית מאפשרים counterstaining axonal. שני הפרוטוקולים מתאימים לדמיין לוקליזציה axonal של Atf4 mRNA במו הבוגרלהשתמש ומוח אנושי.

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			<td>custom probe targeting residues 20-1381 of the mouse Atf4 mRNA (NM_009716)</td>
			<td>Advanced Cell Diagnostics</td>
			<td>-</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>custom probe targeting residues 15-1256 of the human ATF4 mRNA (NM_001675.2)</td>
			<td>Advanced Cell Diagnostics</td>
			<td>-</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>negative control probe-DapB</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">310043</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>positive control probe-mouse Polr2A (optional)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">312471</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>positive control probe-human PPIB (optional)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">313901</td>
			<td>probe</td>
		</tr>
		<tr>
			<td>RNAscope Fluorescent Multiplex Reagent Kit (for fluorescence detection)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">320850</td>
			<td>In situ hybridization kit</td>
		</tr>
		<tr>
			<td>RNAscope 2.0 HD Reagent Kit - BROWN (for chromogenic detection)</td>
			<td>Advanced Cell Diagnostics</td>
			<td align="right">310035</td>
			<td>In situ hybridization kit</td>
		</tr>
		<tr>
			<td>Goat polyclonal anti-ChAT antibody</td>
			<td>Millipore</td>
			<td>AB144P</td>
			<td></td>
		</tr>
		<tr>
			<td>Luxol Fast Blue-Cresyl Echt Violet Stain Kit</td>
			<td>American MasterTech</td>
			<td>KTLFB</td>
			<td></td>
		</tr>
		<tr>
			<td>Clearify clearing agent (xylene substitute)</td>
			<td>American MasterTech</td>
			<td>CACLEGAL</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold mounting&#160; medium with DAPI</td>
			<td>Life Technologies</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX mounting medium</td>
			<td>Sigma</td>
			<td align="right">6522</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of axonally localized mrnas in brain sections using high-resolution in situ hybridization

mRNAs לעתים קרובות מקומי לאקסונים חוליות והתרגום המקומי שלהם נדרש לpathfinding האקסון או הסתעפות בפיתוח ותחזוקה, תיקון או ניוון של מערכת עצבים בתקופות postdevelopmental. ניתוחי תפוקה גבוהים חשפו לאחרונה כי יש לי האקסונים transcriptome יותר דינמית ומורכבת יותר מהצפוי בעבר. ניתוח אלה, עם זאת כבר נעשה בעיקר בנוירונים בתרבית שבו ניתן לבודד האקסונים מתאי somato-הדנדריטים. זה כמעט בלתי אפשרי להשיג בידוד כזה ברקמות שלמות in vivo. לפיכך, על מנת לאמת את הגיוס של mRNAs ורלוונטי הפונקציונלי שלהם בכל בעלי חיים, צריכים להיות משולבים באופן אידיאלי ניתוחי transcriptome עם טכניקות המאפשרות ההדמיה של mRNAs באתר. לאחרונה, טכנולוגיות ISH רומן המזהות RNAs ברמת מולקולה בודדת פותחו. זה חשוב במיוחד בעת ניתוח לוקליזציה subcellular של mRNA, RNAs המקומי מאז נמצא בדרך כלל ברמות נמוכות. כאן אנו מתארים שני פרוטוקולים לצורך זיהוי של mRNAs axonally-מקומי באמצעות טכנולוגית RNA ISH רומן רגישה. יש לנו שילוב RNAscope ISH עם counterstain axonal באמצעות אימונוהיסטוכימיה הקרינה או צבעים היסטולוגית כדי לאמת את הגיוס של Atf4 mRNA לאקסונים in vivo בעכבר הבוגר והמוח אנושי.

סיכום קצר של ההליכים שתוארו לעיל מוצג באיור 1. זיהוי אופטימלי של Atf4 mRNA גרגירים באקסונים כולינרגית באמצעות הסרת מסכות מושרה חום כאשר הערכת לוקליזציה axonal של mRNAs, זה הוא קריטי כדי ל?...

Watch this Scientific Journal Video about Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization at JoVE.com

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.

איתור של Axonally המותאם למקום mRNAs בסעיפי מוח באמצעות ברזולוציה גבוהה<em&gt; באתר</em&gt; הכלאה

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

detection-axonally-localized-mrnas-brain-sections-using-high

Research

JoVE Journal

Neuroscience

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

11.7K Views.  Columbia University. 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.

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

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.

פעמיים פלואורסצנטי באתרו הכלאה בסעיפים המוח טריים

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

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

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

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

A combination of different techniques to maximize data collection from mouse tissue is presented.

שילוב של צבע lipophilic, באתרו</emהכלאה>, אימונוהיסטוכימיה ו היסטולוגיה

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

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.

איתור של הגנום והתמלילים של וירוס ה-DNA מתמיד ברקמות עצבית על ידי פלורסנט באתרו הכלאת שילוב עם 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 ...

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.

חוט השדרה מלא פגיעה ומוח Dissection פרוטוקול Wholemount לאחר<em&gt; באתר</em&gt; הכלאה בזחל ים צמד ים

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

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

ברזולוציה גבוהה<em&gt; In vivo</em&gt; פרוטוקול פילוח ידני לאדם בהיפוקמפוס תת תחומים באמצעות הדמיה בתהודה מגנטית 3T

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

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.

מהיר<em&gt; באתר</em&gt; הכלאה באמצעות oligonucleotide בדיקות במוח-התחילית Paraformaldehyde של חולדות עם סרוטונין תסמונת

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.

שילוב קרינה זוגית<em&gt; באתרו</em&gt; כלאה עם Immunolabelling עבור איתור של הביטוי של שלושה גנים בסעיפי מוח עכבר

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

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.

לוקליזציה של גנים קולטן Odorant ב אנטנות ארבה על ידי RNA<em&gt; באתרו</em&gt; הכלאה

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

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

זיהוי של דופמין קולטן D1-אלפא בתוך מכרסמים גרעין האקומבנס על ידי טכנולוגיה חדשנית RNA בחיי עיר זיהוי

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

ברזולוציה גבוהה 3D הדמיה של זיהום וירוס כלבת ברקמת המוח נקי ממס

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...