وأيد هذا العمل من قبل المعاهد الوطنية الأميركية للصحة منح HL082798 HL111580 و.

1. أقسام الكلى الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم قطع الثابتة الفورمالين (10٪) أقسام الكلى جزءا لا يتجزأ من البارافين على شرائح المجهر في سمك 5 ملم باستخدام ميكرومولار HM 355 S. ويمكن تخزين الشرائح لعدة أشهر قبل التحليل. </li></ol> 2. إعداد الحل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحضير 1 لتر من برنامج تلفزيوني 1X في DDH 2 O في أعلى زجاجة المسمار autoclavable. ملء زجاجة أخرى مع 1 لتر من DDH 2 O. إضافة 1 مل من DEPC إلى كل زجاجة، وغطاء ويهز بقوة. السماح للحلول الجلوس بين عشية وضحاها في درجة حرارة الغرفة لتدمير RNAses ثم الأوتوكلاف. إضافة 400 مل توين-L 20-1 من برنامج تلفزيوني 1X لجعل PBS-T. </li><li style=";text-align:right;direction:rtl"> إعداد بروتين K العازلة (50 mMTris، حمض الهيدروكلوريك، ودرجة الحموضة 8.0 و 1 مم CaCl 2 في DEPC المياه المعالجة). </li><li style=";text-align:right;direction:rtl"> تحضير محلول 0.2٪ الجلايسين في برنامج تلفزيوني-T. </li></ol> 3. إعداد الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد كمية كبيرة من العازلة التهجين (50-100 مل) تتألف من 50٪ الفورماميد، 5X SSC، 0.1٪ توين، 9.2ملي حامض الستريك لتعديل درجة الحموضة إلى 6، 50 ميكروغرام / مل الهيبارين، و 500 ميكروغرام / مل الحمض النووي الريبي الخميرة في DEPC معاملة ده 2 O. تقسم إلى 1 مل مأخوذة وتخزينها في -80 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> إزالة البارافين من الأنسجة عن طريق غسل 3X في الزيلين الطازجة لمدة 5 دقائق لكل منهما. </li><li style=";text-align:right;direction:rtl"> ترطيب أقسام الأنسجة بنسبة 5 دقيقة حضانات في خفض تركيزات الإيثانول (100٪، 75٪، 50٪، و 25٪) في DDH 2 O. </li><li style=";text-align:right;direction:rtl"> علاج الشرائح مع ما يكفي من DEPC لتغطية كامل أقسام الأنسجة (حوالي 0.4-0.5 مل) لمدة 1 دقيقة. جعل متأكد من الأنسجة لا تجف. </li><li style=";text-align:right;direction:rtl"> شطف الشرائح في DEPC معاملة PBS-T مرتين لمدة 5 دقائق، وجمع النفايات المحتوية DEPC للتخلص السليم. جميع الكواشف يجب DEPC المعالجة للقضاء على RNAses من هذه النقطة. </li><li style=";text-align:right;direction:rtl"> Prewarm بروتين K العازلة وإضافة بروتين K إلى تركيز النهائي من 10 ميكروغرام / مل. تغطية أقسام الأنسجة مع الحل K بروتين واحتضان عند 37 درجة مئوية لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> بسرعة إزالة proteinasه K الحل وإضافة 0.2٪ الجلايسين في برنامج تلفزيوني-T لمدة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> شطف الشرائح في DEPC معاملة PBS-T مرتين لمدة 30 ثانية لكل منهما. </li><li style=";text-align:right;direction:rtl"> إصلاح المقاطع في تقدم طازجة بارافورمالدهيد 4٪ لمدة 10 دقيقة. </li></ol> 4. تهجين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر من العازلة التهجين (50٪ الفورماميد، 5X SSC، 0.1٪ توين، 9.2 ملي حمض الستريك لتعديل درجة الحموضة إلى 6، 50 ميكروغرام / مل الهيبارين، 500 ميكروغرام / مل الحمض النووي الريبي الخميرة) في قسم الأنسجة، وتغطي مع ريبونوكلياز HybriSlips مجانا مجرد قطع أكبر من أقسام الأنسجة. </li><li style=";text-align:right;direction:rtl"> أقسام Prehybridize في الرطوبة تسيطر فرن التهجين لمدة 2 ساعة في درجة حرارة التهجين يحدد ل &#39;التحقيق 3 المسمى digoxigenin (عادة الحمض النووي تيم لجنة التحقيق -21 ° C، (أي 54 درجة مئوية لمدة مير 382، تحقيق DNA تيم = 75 ° C )، وذلك باستخدام مناديل مختبر الأنسجة غارقة في 50٪ formamide/50٪ 5X SSC للحفاظ على ترطيب الغرفة. </li><li style=";text-align:right;direction:rtl"> تمييع التحقيق ميرنا، والسيطرة الايجابية (U6، RNA النووية الصغيرة) والسيطرة السلبية (تدافعتتسلسل) إلى 40 نانومتر في المنطقة العازلة التهجين (75 ميكرولتر من العازلة هو مطلوب في القسم). </li><li style=";text-align:right;direction:rtl"> تسخين التحقيق في المخزن التهجين عند 65 درجة مئوية لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> إزالة الشرائح من الفرن التهجين وإزالة coverslips والعازلة التهجين الزائدة من prehybridization. </li><li style=";text-align:right;direction:rtl"> إضافة 75 ميكرولتر من العازلة التي تحتوي على التحقيق في قسم الأنسجة، مباشرة إلى الأنسجة. تغطية الأنسجة مع HybriSlips فقط كبيرة بما يكفي لتغطية أقسام الأنسجة. </li><li style=";text-align:right;direction:rtl"> حفظ الشريحة مستوى حامل، والعودة إلى الفرن التهجين للحضانة بين عشية وضحاها في درجة حرارة من الخطوة 4.2 (حوالي 16 ساعة). </li></ol> 5. التشدد غسل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 200 ميكرولتر 2X SSC، تسخينها إلى درجة حرارة التهجين، فقط تحت حافة واحدة لل coverslips للمساعدة في الافراج ساترة من الأنسجة. إزالة ساترة عن طريق إمالة الشريحة. </li><li style=";text-align:right;direction:rtl"> تغسل الشرائح في 200 ميكرولتر من 50٪ الفورماميد، 50٪ 2X SSC في التهجين 3X درجة الحرارة لمدة 30 دقيقة لكل منهما. </li><li style=";text-align:right;direction:rtl"> شطفالشرائح في برنامج تلفزيوني 5X-T في درجة حرارة الغرفة لمدة 5 دقائق كل يوم شاكر المداري على سرعة منخفضة. </li></ol> 6. كشف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احتضان الشرائح في عرقلة العازلة (2٪ الماعز أو مصل الحصان، 2 ملغ / مل في برنامج تلفزيوني BSA-T) لمدة 1 ساعة في درجة حرارة الغرفة على شاكر المداري على سرعة منخفضة. </li><li style=";text-align:right;direction:rtl"> تمييع شظايا مكافحة DIG-AP فاب في عرقلة العازلة في 1:100. إضافة 100 ميكرولتر في قسم الأنسجة، وتغطي مع HybriSlips مجرد قطع كبيرة بما يكفي لتغطية هذا الباب. </li><li style=";text-align:right;direction:rtl"> احتضان الضد في غرفة ترطيب عند 4 درجات مئوية خلال الليل (حوالي 16 ساعة). </li><li style=";text-align:right;direction:rtl"> تغسل الشرائح في برنامج تلفزيوني-T 7X، لمدة 5 دقائق كل يوم شاكر المداري على سرعة منخفضة. </li><li style=";text-align:right;direction:rtl"> تغسل الشرائح العازلة في AP (100 ملي TrisHCl درجة الحموضة 9.5 و 50 ملي MgCl 2، 100MM كلوريد الصوديوم، 0.1٪ توين 20) 3X، لمدة 5 دقائق لكل منهما. </li><li style=";text-align:right;direction:rtl"> إضافة الحل NBT / BCIP إلى عازلة AP (200 μl/10 العازلة مل) </li><li style=";text-align:right;direction:rtl"> إضافة 400-500 مل من المخفف حل NBT / BCIP إلى كل شريحة لتغطية كامل جميع أقسام الأنسجة. تطوير في الظلام، ومستوى، humidifغرفة العبوات الناسفة لمدة 4-5 ساعة. </li></ol> 7. تصاعد الشرائح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يذوى المقاطع في زيادة تركيزات الإيثانول (25٪، 50٪، 75٪، 100٪) لمدة 5 دقائق لكل منهما. </li><li style=";text-align:right;direction:rtl"> احتضان في 5X الزيلين من أجل مسح المقاطع. </li><li style=";text-align:right;direction:rtl"> جبل الشرائح في Permount تصاعد المتوسطة وبين عشية وضحاها الجافة. </li></ol>

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ليس هناك شيء في الكشف عنها.

كان الهدف من هذه المقالة لوصف بروتوكول لميرنا التهجين في الموقع الذي يعمل بشكل جيد في أنسجة الكلى الفورمالين الثابتة. بينما كان يعمل خارج هذا البروتوكول وقد تم تحديد العديد من المصادر المهمة للقطعة أثرية تلطيخ. يمكن الاهتمام الدقيق لهذه النقاط تساعد على تجنب تل...

الرنا الميكروي صغيرة (حوالي 22 النيوكليوتيدات طويلة) غير المكودة الرنا التي يتم إنتاجها التطور الطبيعي. أنها تعمل عادة لقمع بروتين تعبير من خلال القمع متعدية أو تدهور مرنا. miRNAs ربط لأهداف مرنا مع التكامل غير مكتملة، مما يجعل من الممكن لميرنا واحدة لقمع أهداف متعددة. فهم أي نوع من أنواع الخلايا والهياكل التعبير miRNAs هو جزء مهم من فهم الآليات التي من خلالها تغيرات في الخلية ميرنا التعبير وتأثير الظواهر الأنسجة. حين طرق مثل ميرنا التسلسل، QPCR والنشاف الشمالية يمكن استخدامها للكشف لل miRNAs في الأنسجة كله، لا يسمح هذا النهج واحد لتحديد أي نوع من الخلايا المحددة التي جاءت من داخل نسيج معين. تشريح المكونات الخلوية والهيكلية قبل التحليل باستخدام هذه الأساليب يمكن أن يكون صعبا للغاية والظروف اللازمة لتحقيق العزلة الكافية يمكن أن تؤدي إلى alterations في التعبير الجيني أو تدهور الرنا. الرنا الميكروي في الموقع التهجين هي الطريقة المستخدمة لتصور موقع الرنا الميكروي ومستويات التعبير في الأنسجة. هذا الأسلوب هو قيمة خاصة في الأنسجة تتكون من هياكل غير المتجانسة، مثل الكلى. وقد ثبت MicroRNAs للعب دور تنظيمي في 2،3 التنمية الكلى ووظائف الأعضاء 4. كما تبين التعديلات التعبير microRNA أن تشارك مع أمراض الكلى مثل التليف 5-10، اعتلال الكلية السكري 7، سرطان الكلى 11،12، وإصابة الكلى الحاد 13. في بحثنا، وجدنا أن تحسين الرنا الميكروي الموقع التهجين للأنسجة الكلى في كان قيمة لتحديد المواقع الهيكلية الدقيق ميرنا التعبير في كل من الصحة والمرض 14. تحديد التعبير أنبوبي والخلوية من microRNAs مختلفة أمر مهم لأن تنظيمها من تاقد يكون rgets تعتمد على الوظائف الخلوية. في الدول المريضة من المهم أيضا لتحديد كيفية إحداث تغييرات في التعبير ميرنا قد تؤثر على وظيفة. وكان الهدف من الطريقة الموصوفة هنا للبناء على منهجيات ISH القائمة التي وضعتها Kloosterman آخرون 15، 16،17 المحققين الآخرين، وتلك التي اقترحتها Exiqon 1 و تحسين طريقة لأنسجة الكلى الفورمالين الثابتة. وقد استخدمنا بنجاح هذا الأسلوب لتحديد الاختلافات الإقليمية المتميزة في الكلى التعبير الرنا الميكروي مير-382 مع جانب واحد انسداد الحالب 18. ويمكن استخدام هذا النهج مع الأنسجة الأخرى كذلك، مع التحسين إضافية.

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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P6148</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:137px;">70011044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">D5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Tween-20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Xylenes</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">534056</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Ethanol</td>
			<td style="width:71px;">Ultra Pure</td>
			<td style="width:137px;">200CSPTP</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Tris-HCl</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15506-017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2536</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Glycine</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4059-00</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Citric Acid</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2404</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Formamide</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">20x SSC Buffer</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15557-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Heparin</td>
			<td style="width:71px;">SAGENT</td>
			<td style="width:137px;">25021-400-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Yeast RNA</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">AM7118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">M8266-1004</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">NaCl</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4058-01</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Proteinase K</td>
			<td style="width:71px;">Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- miRNA probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">miR dependent-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- Scrambled miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99004-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">3&#8217; DIG- U6 miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99002-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Anti-Digoxigenin-Ab Fab</td>
			<td rowspan="2" style="width:71px;">Roche</td>
			<td rowspan="2" style="width:137px;">11093274910</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Fragments</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NBT/BCIP</td>
			<td style="width:71px;">Roche</td>
			<td style="width:137px;">11681451001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Permount</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">SP15-500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Coverslips</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">Fit to tissue size</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Microtom HM 355 S</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:137px;">23-900-672</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">In-slide Out Hybridization Oven</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">241000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Aluminum Tray Assembly</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403973</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Stainless Steel Rack Insert</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403754</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

microrna in situ hybridization for formalin fixed kidney tissues

في هذه المادة ونحن تصف طريقة للكشف اللونية من ميرنا في الكلى من خلال الموقع التهجين مع digoxigenin في المفتاحية تحقيقات الرنا الميكروي. هذا البروتوكول، وضعت أصلا من قبل Kloosterman وزملاؤه للاستخدام واسع النطاق مع تحقيقات Exiqon ميرنا 1، تم تعديل للتغلب على التحديات الكامنة في تحليل ميرنا في أنسجة الكلى. وتشمل هذه القضايا مثل تحديد هيكل والصعب إزالة التحقيق المتبقية والأجسام المضادة. استخدام رقيقة نسبيا، 5 مم، أقسام الأنسجة يسمح لتصور واضح لهياكل الكلى، في حين تم الإبقاء على إشارة قوية التحقيق في الخلايا. بالإضافة إلى ذلك، تم تحسين ظروف الاعتقال والتحقيق الحضانة لتسهيل التصور والتعبير microRNA مع انخفاض الخلفية وإشارة غير محددة. هنا، يتم وصف بروتوكول الأمثل، الذي يغطي مجموعة الأنسجة الأولية وإعداد من خلال تركيب الشرائح في نهاية الإجراء. وcompone الأساسيةاليلة من هذا البروتوكول يمكن تغييرها لتطبيقها على الأنسجة الأخرى ونماذج ثقافة الخلية.

مجالات قسم الأنسجة التي تصبح جفت خلال التهجين، حضانات أو غسل الخطوات عموما في نهاية المطاف أكثر تلطيخ بحزن خلال تطوير NBT / BCIP. الشكل 1 يبين جزء من قسم الكلى الذي تراجع HybriSlip الخروج من حافة الأنسجة، والسماح لها لتصبح المجففة جزئيا. على الرغم الإماهة والتغطية في ...

Watch this Scientific Journal Video about MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues at JoVE.com

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

توضح هذه المقالة في البروتوكول الموقع التهجين الأمثل للكشف colormetric التعبير الرنا الميكروي في الفورمالين أقسام الكلى ثابتة.

الرنا الميكروي<em&gt; في الوضع الطبيعي</em&gt; التهجين للأنسجة الكلى الفورمالين الثابتة

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

microrna-in-situ-hybridization-for-formalin-fixed-kidney-tissues

Research

JoVE Journal

Biology

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

14.8K Views.  Medical College of Wisconsin.  توضح هذه المقالة في البروتوكول الموقع التهجين الأمثل للكشف colormetric التعبير الرنا الميكروي في الفورمالين أقسام الكلى ثابتة. 

Video: MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

الجامعة جبل مزدوجة في الموقع التهجين لافراخ الدجاج في وقت مبكر

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

الزرد الجامع جبل الرفيع قرار مزدوج نيون في الموقع التهجين

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

مضان في الموقع التهجين (FISH) البروتوكول في الحيوانات المنوية البشرية

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

في الموقع التهجين لتوطين دقيقة من النصوص في النباتات

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

المشعة في الموقع التهجين، لتحديد متنوعة أنماط التعبير الجيني في الأنسجة

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

الكشف عن الحمض النووي الريبي الفيروسي بواسطة الإسفار<em&gt; في الموقع</em&gt; التهجين (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

تحليل النسخ الجيني وحيدة الخلية التي كتبها نيون RNA<em&gt; في الموقع</em&gt; التهجين (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

التوقيت كروموسوم النسخ المتماثل جنبا إلى جنب مع نيون<em&gt; في الموقع</em&gt; التهجين

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

كله جبل RNA نيون<em&gt; في الموقع</em&gt; التهجين من<em&gt; ذبابة الفاكهة</em&gt; الأجنة

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

التصور والتحليل من جزيئات الرنا المرسال عن طريق الإسفار<em&gt; في الموضع</em&gt; التهجين في<em&gt; خميرة الخباز</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...