نشكر N. ساوتيل (الأطفال في سينسيناتي المركز الطبي في مستشفى سينسيناتي، أوهايو، الولايات المتحدة الأمريكية)، S. افستاتيو (جامعة كامبريدج، المملكة المتحدة) وجيمس هيل (مركز العلوم الصحية LSU، نيو أورلينز، الولايات المتحدة الأمريكية) لتقديم عينات من HSV-1 الفئران والأرانب المصابة، على التوالي، وللمواد؛ H. ماسوموتو (معهد بحوث Kazusa الحمض النووي، شيبا، اليابان) وS. Khochbin (معهد ألبرت Bonniot، غرونوبل، فرنسا) لإجراء مناقشات مفيدة. وقد تم تمويل هذا العمل من المنح المقدمة من المركز الوطني للبحوث العلميه (CNRS) (برنامج ATIP، إلى PL، http://www.cnrs.fr)، والوكالة الفرنسية القومي للبحوث (وكالة الاستخبارات الوطنية) (ANR-05-MIIM- 008-01، CENTROLAT، <a href="http://www.agencenationale-recherche.fr">http://www.agencenationale-recherche.fr</a> )، ومؤسسة FINOVI ( <a href="http://www.finovi.org/:fr:start">http://www.finovi.org/:fr:start</a> )، وLabEX DEVweCAN (ANR-10-LABX-61 ) من جامعة دي ليون، في إطار برنامج &quot;Investissemenنهاية الخبر كوت أفينير &quot;(ANR-11-0007-آيدكس) التي تديرها وكالة الاستخبارات الوطنية ( <a href="http://www.agence-nationale-recherche.fr">http://www.agence-nationale-recherche.fr</a> )، أنا لا تصب جمعية بحوث السرطان مناهضة جنيه (ARC-ARC-7979 و 4910، <a href="http://www.arc-cancer.net">http://www.arc-cancer.net</a> )، الدوري الفرنسي الوطني كونتر جنيه السرطان (هروب، <a href="http://www.ligue-cancer.net">http://www.ligue-cancer.net</a> )، والإنكا (برنامج EPIPRO، <a href="http://www.e-cancer.fr">http://www.e -cancer.fr</a> ). FC وPL هم باحثون CNRS.

الكتاب تعلن أي المصالح المالية المتنافسة.

بروتوكول الموصوفة هنا يسمح للكشف عن HSV-1 الجينوم الكامنة داخل الخلايا العصبية من الماوس أقسام الأنسجة العصبية. وقد فهمنا من مسارات تنظيم التعبير الجيني الفيروسي محدودة بسبب عدم وجود طريقة للكشف عن HSV-1 الحمض النووي الجيني في الموقع الطبيعي داخل الأنسجة العصبية. و...

<html> نوع فيروس الهربس البسيط 1 (HSV-1) هو فيروس موجه للعصب الإنسان الثابتة، وإقامة العدوى الكامنة على المدى الطويل في الخلايا العصبية في العقد الثلاثي التوائم (TG) في الجهاز العصبي المحيطي، والتي كانت تعيد تجديد دوري لتكرار وانتشار. الجينوم HSV-1 هو 150 كيلو بايت dsDNA وإضفاء الطابع المحلي في نواة الخلايا العصبية المضيفة حيث لا يزال البلازميدات chromatinized multicopy كما، والتي لا الاندماج في الجينوم الخلية المضيفة 1،2. خلال الكمون، وقمعها بشدة برنامج جيني HSV-1 دورة تنسخي، ويقتصر التعبير الجيني للنسخة المرتبطة الكمون (LAT) موضع، من إنشاء الكمون لبدء تنشيط 3. LAT تنتج 8.5 كيلو بايت طويلة غير المكودة RNA معالجتها في كبرى 2 كيلو بايت الوهق مستقرة، والعديد من ميرنا 4-7. وهكذا يتميز HSV-1 كمون من خلال وجود الحمض النووي الفيروسي الجيني، LAT الحمض النووي الريبي، وعدم وجود كشف البروتينات دورة تنسخي. ontent &quot;&gt; النماذج الحيوانية، في الغالب الماوس والأرنب، هي نماذج تجريبية تلخص العديد من الميزات من الكمون في الإنسان. واحدة من المصالح الرئيسية لتلك النماذج هو أنها تسمح بدراسة الجوانب الفسيولوجية للHSV-1 كمون في المضيفين مناعيا. على مدى العقود الماضية ، العديد من الأدوات التجريبية، مثل الفيروسات والفئران المعدلة وراثيا، وقد وضعت لدراسة علم وظائف الأعضاء، وعلم الوراثة، وعلم الأحياء الخلوية من HSV-1 كمون، من الأنسجة الحيوانية. وحتى الآن، تم الكشف عن الحمض النووي الجيني الفيروسي وكميا بواسطة لطخة الجنوبية و QPCR من فصلها TGS. ومع ذلك، لا يوجد حاليا أي وسيلة متاحة للكشف عن HSV-1 الجينوم بواسطة التهجين في الموقع على أقسام الأنسجة 8، وبالتالي يتم تقييم الكمون بشكل روتيني على المقاطع النسيجية من خلال الكشف عن الحمض النووي الريبي RNA LAT بواسطة التهجين في الموقع بدلا من الفيروسية كشف الجينوم. لأنه قد كان من المستحيل أن تميز الخلايا المصابة على أساس وجود الجينوم الفيروسي، ثيوقد ق الحد التقنية والعيب الرئيسي لتحليل جوانب كثيرة من التفاعلات المضيفة للفيروسات، مثل العلاقة بين الجينوم الفيروسي والخلوية والفيروسية التعبير الجيني أو بوساطة الخلية المضيفة استجابة مناعية 9،10. الأهم من ذلك، عدم التجانس الخلية الى خلية من العدوى الكامنة لا تزال غير مستكشفة نسبيا، ولقد ثبت أن تكون سمة رئيسية من الكمون في الفئران في الخلايا العصبية والحسية العقدة الإنسان زرعها في الفئران SCID 11-17. عادة، فقد تبين من خلال QPCR أن الجينوم عدد النسخ HSV-1 في الخلية يختلف من 5 إلى عدة مئات. على الرغم من أن LAT يبدو كمنظم رئيسي من الكمون وتنشيط، أشارت البيانات QPCR على الخلايا العصبية معزولة في الموقع وPCR أنه ليس هناك سوى مجموعة فرعية من الخلايا العصبية المصابة خفية، منخفضة تصل إلى 30٪، ويعرب عن موضع LAT 11،12،18-21. كيف الخلية المضيفة والبيئة الخلوية داخل الأنسجة تأثير على إنشاء الكمون الفيروسات ويبقى التعبير الجيني الفيروسي غير واضحة. نحن هنا وصف الفلورسنت قوة التهجين الموضع (FISH) طريقة للكشف الفعال للنسخ منخفضة HSV-1 الحمض النووي الجيني داخل أقسام الأنسجة العصبية في الحيوانات. وقد تم تصميم هذه الطريقة واستخدامها من قبلنا للوصول إلى عالية الدقة التصوير المجهري ما هو ضروري لدراسة تفاعل الجينوم الفيروسي مع الخلية المضيفة المكونات داخل النووية 22. بالإضافة إلى ذلك، نحن تصف طريقة تلوين متعددة للكشف في وقت واحد من الحمض النووي الفيروسي RNA والبروتينات مع، التي هي أداة فريدة لوصف التفاعلات الفيروسات المضيف التي تنظم التعبير الجيني الفيروسي. ويمكن أيضا أن تطبق طريقة لمجموعة واسعة من التحليلات التي تتطلب الكشف عن HSV-1 الجينوم الكامنة، مثل قياس الخلايا العصبية المصابة في عدد كبير من الأقسام. والخطوة الرئيسية هي تطبيق المستضد العلاج استرجاع لجعل الحمض النووي الفيروسي للوصول إلى التهجين. وبالتالي، قد يكون هذا البروتوكول أيضا فعالة لكشفمن الفيروسات dsDNA وغيرها، والتي هي حاليا لا يمكن كشفها بواسطة النهج الحمض النووي FISH التقليدية داخل الأنسجة الحيوانية.

<table border="0" cellpadding="0" cellspacing="0" width="822">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:203px;">Balb/c mice</td>
			<td style="width:189px;">Janvier, France</td>
			<td style="width:93px;"> </td>
			<td style="width:337px;">6 week-old females</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;">HSV-1 strains</td>
			<td>SC16 strain (wild type)</td>
			<td></td>
			<td style="width:337px;">See Labetoule, M. et al. (2003) Invest Ophthalmol Vis Sci 44: 217&#8211;225, for details on HSV-1 strain and virus stock preparation.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ketamine hydrochloride</td>
			<td>Sigma</td>
			<td>K2753</td>
			<td rowspan="2" style="width:337px;">Intraperitoneal injection of a solution containing Ketamine (100mg/kg) and Xylazine (10mg/kg)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazine hydrochloride</td>
			<td>Sigma</td>
			<td>X1251</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>158127</td>
			<td style="width:337px;">Suspend 4g of PFA in 90mL of water. Add 50&#181;L of 1N NaOH, and heat at 60&#176;C in a water bath with agitation. PFA dissolves in about 30min. Add 10mL of 10X PBS. This solution can be prepared in advance and stored at -20 &#176;C in 5mL tubes. Caution. Manipulate under a fume hood.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Physiological Saline</td>
			<td>Sigma</td>
			<td colspan="2">07982-100TAB-F</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBS, pH 7.4 (sterile)</td>
			<td>Life Technologies</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sucrose</td>
			<td>Sigma</td>
			<td>84100</td>
			<td>Prepare a 20% sucrose solution in 1X PBS.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Cryosectionning embedding medium - Tissue-Tek OCT Compound -</td>
			<td>SAKURA</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Large vector DNA purification kit</td>
			<td>Qiagen</td>
			<td>12462</td>
			<td style="width:337px;">To purify Cosmid or BAC vector containing HSV-1 genome and store at -20&#176;C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Nick translation kit</td>
			<td>Roche Applied Sciences</td>
			<td>10 976 776 001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cy3-dCTP</td>
			<td>GE Healthcare</td>
			<td>PA53021</td>
			<td>Protect from light</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">0.5M EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">G50 Mini spin column</td>
			<td>GE Healthcare</td>
			<td>27-5330-01 </td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Salmon sperm DNA 10mg/mL</td>
			<td>Invitrogen / Life Technologies</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol molecular biology grade</td>
			<td>Sigma</td>
			<td>87047</td>
			<td>Prepare a 70% solution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Salmon sperm DNA</td>
			<td>Invitrogen / Life Technologies</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formamid Molecular biology grade</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td>Caution. Manipulate under fume hood.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HSV-1 biotinylated commercial probe </td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-40838</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ImmEdge hydrophobic pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">20X Saline Sodium Citrate (SSC)</td>
			<td>Sigma</td>
			<td>S6639</td>
			<td>Prepare a 2X SSC solution in ddH20.</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td style="width:337px;">Prepare a 10% stock solution in water and store at +4&#176;C. Prepare the 0.5% solution in 1X PBS right before use.</td>
		</tr>
		<tr height="102">
			<td height="102" style="height:102px;">10mM sodium citrate pH 6.0</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td style="width:337px;">Prepare a 100mM stock solution (10X). Weigh 10,5g of citric acid (MW 210.14. Caution, irritant and toxic, wear appropriate mask and gloves), and dissolve in 400mL water. Adjust pH at 6.0 with 1N NaOH (caution, irritant, wear gloves). Adjust to 500mL with distilled water. Dilute 10 times in distilled water before use.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acetic Acid</td>
			<td>Sigma</td>
			<td>320099</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Methanol, molecular biology grade</td>
			<td>Sigma</td>
			<td>322415</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dextran sulfate - MW 500 000</td>
			<td>Euromedex</td>
			<td>EU0606-A</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Denhardt&#39;s solution (100X)</td>
			<td>Euromedex</td>
			<td>1020-A</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rubber Cement &#34;FixoGum&#34;</td>
			<td>Marabut</td>
			<td>290110000</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">DNA purification kit - Qiaquick PCR purification kit -</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T7 in vitro transcription kit</td>
			<td>Ambion / Life Technologies</td>
			<td>AM1314</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Biotin-16-UTP</td>
			<td>Roche Applied Sciences</td>
			<td>11388908910</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNA purification mini-column</td>
			<td>Qiagen</td>
			<td>73404</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ribonucleoside Vanadyl Complex</td>
			<td>New England Biolabs</td>
			<td>S1402S</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">H2O2</td>
			<td>Sigma</td>
			<td>H3410</td>
			<td style="width:337px;">Prepare a 3% solution in distilled water. Store at +4 &#176;C and protect from light.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401011</td>
			<td style="width:337px;">prepare a 10mg/mL solution in RNAse free water</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Normal Goat Serum</td>
			<td>Invitrogen</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">Primary antibodies</td>
			<td>Any supplier</td>
			<td></td>
			<td style="width:337px;">The following primary antibodies were used in the result section: anti-mouse CENP-A (rabbit mAb C51A7, Cell Signaling Technologies), and anti-ATRX H-300 (Santa Cruz Biotechnology)</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Secondary fluorescent antibodies</td>
			<td>Invitrogen / Life Technologies</td>
			<td></td>
			<td style="width:337px;">The fluorescent secondary antibodies routinely used in our protocol are AlexaFluor labeled goat antibodies (IgG H+L). The antibody used in the result section is an anti-rabbit goat antibody labaled with AlexaFluor 488 (reference A11001)</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Tyramide Signal Amplification (TSA) kit - Streptavidin + AlexaFluor 350 (blue fluorescence)</td>
			<td>Invitrogen / Life Technologies</td>
			<td>#T20937</td>
			<td rowspan="2" style="width:337px;">TSA kits are also available from Perkin Elmer</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Tyramide Signal Amplification (TSA) kit - Streptavidin + AlexaFluor 488 (green fluorescence)</td>
			<td>Invitrogen / Life Technologies</td>
			<td>#T20932</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Hoechst 33342</td>
			<td>Invitrogen / Life Technologies</td>
			<td>H3570</td>
			<td style="width:337px;">Prepare a 0.5&#181;g/mL solution in 1X PBS immediatly before use. Discard the remaining solution.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">22x50mm coverslip. n&#176;1.5 glass.</td>
			<td>Electron Microscopy Sciences</td>
			<td>72204-04</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;"> Mounting medium with anti-fading agent - Vectashield -</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td style="width:337px;">Another conventional product is Fluoromount G from electron microscopy Science</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Superfrost glass slides</td>
			<td>FisherScientific</td>
			<td>12-550-15</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Equipment / material</td>
			<td>Company</td>
			<td>Reference</td>
			<td>Note</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Needle for infection</td>
			<td></td>
			<td></td>
			<td>Glass micropipette hot drawn. Home made.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dissection equipement</td>
			<td>Moria, France</td>
			<td></td>
			<td>Microsurgical scissors and forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Peristaltic pump</td>
			<td style="width:189px;">Cole Palmer Instruments</td>
			<td></td>
			<td>Easyload Masterflex</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro-syringe pump device (Nano Pump)</td>
			<td>kdScientific</td>
			<td>KDS310</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cryostat</td>
			<td>Leica France</td>
			<td>CM 1510-1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">-80 &#176;C freezer</td>
			<td>Sanyo</td>
			<td></td>
			<td>Ultra Low -80&#176;C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Domestic microwave oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dry block heater</td>
			<td>Eppendorf</td>
			<td>022670204</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Incubator Slide moat</td>
			<td>Boekel Scientific</td>
			<td>240000</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coplin Jar</td>
			<td>Dominique Dutscher</td>
			<td>68512</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Staining glass container</td>
			<td>Dominique Dutscher</td>
			<td>68506</td>
			<td></td>
		</tr>
		<tr height="136">
			<td height="136" style="height:136px;">Fluorescent microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td style="width:337px;">The images presented in the result section were collected with a Zeiss AxioObserver with objective x40 LD NeoFluor N.A 0.6, and x100 PlanApochromat N.A 1.3. Filter set #38, #43 and #43. HXP 120 fluorescence light source. Photometrics CoolSNAP HQ2 CCD camera. Signal will be more easily observed on a recent high efficiency microscope such as Zeiss AxioImager/AxioObserver series, Nikon Ti-E/Ni-E series or Leica DM/DMI6000 series</td>
		</tr>
	</tbody>
</table>

detection of the genome and transcripts of a persistent dna virus in neuronal tissues by fluorescent in situ hybridization combined with immunostaining

codetection خلية واحدة من الجين والمنتجات RNA والبروتينات الخلوية التنظيمية أمر بالغ الأهمية لدراسة الجينات تنظيم التعبير. هذا هو التحدي في مجال علم الفيروسات، وبصفة خاصة عن الفيروسات الحمض النووي المستمر-النووية التي تنطوي على تكرار نماذج حيوانية لدراستهم. نوع فيروس الهربس البسيط 1 (HSV-1) يحدد عدوى مدى الحياة كامنة في الخلايا العصبية الطرفية. يقدم الفيروس كامنا كما الخزان، من الذي ينشط ويؤدي الى حلقة جديدة العقبولية. يبقى بيولوجيا الخلية من HSV-1 كمون غير مفهومة، ويرجع ذلك جزئيا إلى عدم وجود طرق للكشف عن HSV-1 الجينوم في الموقع في النماذج الحيوانية. نحن تصف الحمض النووي الفلورسنت الموقع التهجين (FISH) في نهج كشف بكفاءة-نسخة منخفضة الجينوم الفيروسي داخل أقسام الأنسجة العصبية من النماذج الحيوانية المصابة. وتعتمد الطريقة على القائم الحرارة مستضد إماطة اللثام، وتحقيقات الحمض النووي المسمى مباشرة الصنع، أو تحقيقات متاحة تجاريا. وضعنا الفول الثلاثينهج نينغ، والجمع بين الحمض النووي RNA-FISH مع FISH والمناعي، وذلك باستخدام البيروكسيديز مقرها إشارة التضخيم لاستيعاب كل متطلبات تلطيخ. وتحسن كبير هو القدرة على الحصول، في غضون 10 ميكرومتر أقسام الأنسجة، وإشارات منخفضة الخلفية التي يمكن تصويرها بدقة عالية بواسطة المجهر متحد البؤر واسعة المجال epifluorescence التقليدية. بالإضافة إلى ذلك، عملت تلطيخ ثلاثية مع مجموعة واسعة من الأجسام المضادة الموجهة ضد البروتينات الخلوية والفيروسية. بروتوكول كاملة يأخذ 2.5 أيام لاستيعاب الأجسام المضادة والاختراق التحقيق داخل الأنسجة.

بعد عدة شهور من التجارب واسعة النطاق، اكتشفنا أن فضح الكيميائية القائمة على الحرارة الكامنة جعلت HSV-1 الجينوم المتاحة لفلوري التهجين في الموقع. خلال هذه العملية، حاولنا مختلف الإجراءات إماطة اللثام، والعلاجات فقط على أساس الحرارة (أي تسخين أقسام تصل إلى در?...

Watch this Scientific Journal Video about Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining at JoV

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

أنشأنا فلوري التهجين الموضع البروتوكول في للكشف عن فيروس الحمض النووي الجينوم المستمرة داخل أقسام الأنسجة من النماذج الحيوانية. هذا البروتوكول يتيح دراسة عملية العدوى عن طريق codetection من الجينوم الفيروسي، والمنتجات RNA لها، والبروتينات الفيروسية أو الخلوية داخل الخلايا واحد.

الكشف عن الجينوم والنصوص من الفيروسات الحمض النووي في الخلايا العصبية الأنسجة الثابتة التي كتبها نيون في الوضع الطبيعي التهجين جنبا إلى جنب مع المناعية

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

detection-genome-transcripts-persistent-dna-virus-neuronal-tissues

Research

JoVE Journal

Neuroscience

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

18.2K Views.  CNRS UMR 5534.  أنشأنا فلوري التهجين الموضع البروتوكول في للكشف عن فيروس الحمض النووي الجينوم المستمرة داخل أقسام الأنسجة من النماذج الحيوانية. هذا البروتوكول يتيح دراسة عملية العدوى عن طريق codetection من الجينوم الفيروسي، والمنتجات RNA لها، والبرو...

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

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.

نقرا الإسفار في الموقع التهجين في أقسام المخ طازجة

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

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

سريع الصغرى، الرحلان الشاردي من الغلوتامات وGABA: أداة مفيدة لبحث تكامل متشابك

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

حمل اللدونة من نجمي المستقبلات بواسطة التلاعب أسعار اطلاق النار متعلق بالخلايا العصبية

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

حقن الفيروسي Intracerebroventricular من حديثي الولادة ماوس الدماغ للاستمرار وانتشار تنبيغ العصبية

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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.

الكشف عن Axonally المترجمة من mRNAs في أقسام المخ باستخدام عالية الدقة<em&gt; في الموقع</em&gt; التهجين

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

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

الاستفادة المشتركة منهجيات لتحديد دور البلازما غشاء تسليم خلال أكسون التشعب والعصبية التشكل

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

خارج الخلية تسجيل النشاط العصبية جنبا إلى جنب مع Microiontophoretic تطبيق المواد Neuroactive في الفئران مستيقظا

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

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.

الجمع بين نقرا الإسفار<em&gt; في الموقع</em&gt; التهجين مع Immunolabelling للكشف عن التعبير عن ثلاثة جينات في أقسام الدماغ ماوس

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.

توطين الجينات مستقبلات أودورانت في هوائيات الجراد بواسطة الحمض النووي الريبي<em&gt; في الموقع</em&gt; التهجين

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.