We are grateful to the technical support from Ms. Hong Xian Hsing, Mr. Min Jeng Li, and Mr. Eric C. P. Wu. We express our appreciation to Miranda Loney for English editing.

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No conflicts of interest declared.

الخلايا النباتية والجدران الصلبة المحمولة، والألياف صعبة، البلورات، ونسبة عالية من المياه التي تسبب مشاكل تمزق الأنسجة أثناء باجتزاء الأنسجة النباتية. على الرغم من أن كثيرا ما يستخدم باجتزاء القائم على البرافين لأنسجة النبات، وبلورات الذاتية غالبا ما ممزق الأنسجة النباتية خلال باجتزاء (الشكل 1). ونظرا لنسبة عالية من المياه بطبيعتها داخل الخلايا النباتية، باجتزاء القائم ناظم البرد وغالبا ما يسبب خلايا مكسورة وأقسام الأنسجة متصدع. في هذه الدراسة، تم تطوير جنبا إلى جنب بروتوكول البارافين التضمين وcryosection اسمه الهجين قص وتم الحصول عليها جيدة أقسام الأنسجة الجودة. هذا البروتوكول يحل المشكلة المرتبطة نسبة عالية من المياه عن طريق إدخال البارافين التضمين، ويقلل من تأثير تمزق لتصلب شمع البارافين تحت درجة حرارة منخفضة خلال باجتزاء (الشكل 3). لذلك، هذا البروتوكول المعدل هو مفيد أكثر من أي التقطيعة القائم على البرافيننينغ أو cryosectioning للحفاظ على سلامة الأنسجة النباتية. توضح هذه المخطوطة أن طريقة الهجين قص يحافظ على سلامة الأنسجة في العديد من الأنسجة من فالاينوبسيس السحلبية مثل برعم إبطي، والبذور، وPLB، الخ التي تحتوي على مستويات عالية من بلورات (الشكلان 3-4). وعلاوة على ذلك، وهذا البروتوكول هو قابل للمحاصيل الحبوب مثل الأعضاء التناسلية وأوراق الأرز، والذرة، والقمح التي تحتوي على السيليكا عالية (الشكلان 5-6). ويفترض، ويمكن تطبيق هذا البروتوكول لودي النباتات التي تحتوي على الألياف عالية. بشكل عام، وتحديد الأنسجة بدقة بالغة الأهمية لالهجين قص. وجدنا أن تثبيتي حامض الفورمالديهايد الكحول الحمضية (FAA) هو أفضل من منهاج العمل للحفاظ على سلامة الأنسجة من بعض الأنسجة المتمردة مثل براعم محوريا، والجذور، وما إلى ذلك، PFA يعمل بشكل أفضل من القوات المسلحة الأنغولية في الحفاظ على سلامة الحمض النووي الريبي. لذلك يوصى PFA لإصلاحعينة لفي الموقع التهجين (ISH) العمل. تم تصميم البروتوكول الموصوفة هنا لتجربة RNA ISH. لذلك، تم إعداد جميع الكواشف لتجنب تدهور الحمض النووي الريبي عن طريق القضاء على التلوث ريبونوكلياز عن طريق العلاج DEPC. إذا الهجين قص القسم للدراسات تشريحية، التناضح العكسي (RO) المياه العادية وعازلة أو الكواشف المشتقة منها مقبولة. تقليل حجم العينة وسماكة أقل من 3 مم مفيد للتسلل. وعلاوة على ذلك، وزيادة الوقت الغمر للجفاف وتسلل ضرورية لأنسجة الملمس الصعبة. الحد من هذا البروتوكول قد يرجع ذلك إلى المشاكل الناجمة عن عدم كفاية التثبيت، والجفاف، وتسلل من العينة. لذلك، وتعديل الوقت لتجهيز كل خطوة أمر بالغ الأهمية لإنتاج كتلة البارافين نوعية جيدة. عادة، الأنسجة أصعب تحتاج وقتا أطول معالجة من الأنسجة ليونة. وبالإضافة إلى ذلك، أظهرت لنا أن الهجين قص عملت بنجاح في تركيبة وايISH التاسع لتوفير التوزيع المكاني للنصوص مختارة (الشكل 7). باختصار، هذا البروتوكول هو مفيد لدراسة علم تشريح النبات ويوفر خريطة الحمض النووي الريبي الأنسجة محددة للجينات منتقاة. بالإضافة إلى ذلك فإنه يمكن تطبيقها على الدراسات الجزيئية الأخرى مثل محطة deoxynucleotidyl ترانسفيراز dUTP نيك نهاية الوسم (النفق) مقايسة، مضان في الموقع التهجين (FISH)، والتقنيات المناعية. في الختام، هذا المحسن بروتوكول باجتزاء الأنسجة على حد سواء مفيدة ومفيدة للباحثين في المجتمعات النباتية.

طريقة باجتزاء القائم على البرافين هو أسلوب يستخدم على نطاق واسع للدراسات تشريحية. الحفاظ على تشريح الأنسجة سليمة مهم للدراسات المورفولوجية والبيولوجية. تقنية باجتزاء جزءا لا يتجزأ من البارافين هو مفيد لالبارافين التضمين يحتفظ الخلايا والأنسجة التشكل. وبالإضافة إلى ذلك، وكتل البارافين ويمكن تخزين مريح لفترات طويلة من الزمن. ومع ذلك، باجتزاء جزءا لا يتجزأ من البارافين ليست مناسبة للأنسجة النباتات التي تحتوي على بلورات داخل الخلايا. بلورات داخل الخلايا في كثير من الأحيان المسيل للدموع وأشرطة البارافين وسلامة الأنسجة الضرر أثناء باجتزاء. على عكس باجتزاء البارافين، cryosectioning سريع نسبيا ويمكن الحصول على المقاطع دون تثبيت، والجفاف تسلسلي أو تضمين تسلل المتوسط 1. Cryosections متوافقة مع العديد من التطبيقات مثل المناعية والتهجين في الموقع، والكيمياء النسيجية الانزيم. ميزة أخرى من cryosectioning هيأن هذا الأسلوب لا تذهب من خلال عمليات تمسخ المحتملة مثل ارتفاع درجة الحرارة والعلاجات الكيميائية، والجزيئات الخلوية بحيث يتم الاحتفاظ بشكل جيد داخل أقسام الأنسجة 2. ويفضل Cryosectioning عموما على القائم على البرافين باجتزاء للدراسات في الأنسجة الحيوانية. ومع ذلك، cryosectioning ليس هو الخيار الأول في النباتات لدرجة حرارة التجمد يسبب في بعض الأحيان تكوين بلورات الثلج التي تؤثر على نوعية من قسم السلامة. على الرغم من أن التنظيم الاسموزي مثل حلول السكروز، البولي ايثيلين جلايكول (PEG)، أو الجلسرين 3 تم الإبلاغ للحد من تكون الجليد الكريستال في ظل ظروف التجميد، وتحسين أقل من المستوى الأمثل. على التكيف مع بيئات مختلفة، وغالبا ما يكون نباتات مختلفة تطورت متميزة خلايا نسيج نسيج ومصنع لتشكيل الجدران الصلبة المحمولة، والألياف صعبة، وبلورات 4،5. على سبيل المثال، غير قابلة للذوبان بلورات أكسالات الكالسيوم والهيئات السيليكا شائعة إلى حد ما فيمصانع 6. / تم الإبلاغ الهيئات سيليكات بلورات للمساعدة في الحفاظ على بنية النبات، الإنتصاب، والوقاية من الأمراض أو الآفات في نباتات الحبوب 7-9. بساتين الفاكهة بوعاء وقطع زهرة السحلبية سوق مزدهرة وأنه هو الصناعة المتنامية. فالاينوبسيس أفروديت (العثة السحلية) هو واحد من نباتات الزينة التصدير الأكثر أهمية في تايوان. وقد بذلت جهود كبيرة لفهم التغيرات المورفولوجية والفسيولوجية للعمليات المزهرة في بساتين الفاكهة فالاينوبسيس. وبدأت المسامير الزهور من بساتين الفاكهة فالاينوبسيس من البراعم الإبطية في قاعدة ورقة. بعد فترة من درجة الحرارة المحيطة باردة (ما يقرب من شهر ونصف)، براعم الإبطين تكبير، وكسر السكون، وتبرز من قاعدة ورقة على التطور إلى ارتفاع الأزهار الصغيرة. لفهم الفسيولوجية والخلوية، والعمليات الجزيئية للبدء السنبلة، لا بد من تطوير تقنية التشريحية قوية لvisuaالأنسجة ليز أو علامات في الوقت المناسب. ومع ذلك، فإن وجود بلورات في كل مكان في أنسجة السحلية، لا سيما في البراعم الإبطية، ويجعل العمل التشريحية صعوبة. نحن هنا سعت إلى تحسين قسم سلامة الأنسجة النباتية المتمردة التي تعتبر حتى الآن تحديا من الناحية التقنية. نحن هنا تظهر تحسن بروتوكول اسمه الهجين قص. وهي طريقة باجتزاء القائم على البرافين التي يتم تنفيذها باستخدام ناظم البرد. تضمين البارافين يحل نسبة عالية من المياه في الأنسجة النباتية. باجتزاء تحت درجة حرارة منخفضة يصلب كتلة البارافين، ويقلل من وضوح الشمس تمزيق المشكلة، ويحسن سلامة الأنسجة بشكل كبير. هذا البروتوكول بشكل كبير على تحسين سلامة الأنسجة للعينات النباتية المتمردة.

<table border="0" cellpadding="0" cellspacing="0" width="734">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Embedding Center model EC780-1</td>
			<td style="width:127px;">CSA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Microtome model RM2255</td>
			<td style="width:127px;">Leica</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Cryostat model CM 1950</td>
			<td style="width:127px;">Leica</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Axio Scope A1 microscope&#160;</td>
			<td style="width:127px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Murashige &#38; Skoog medium including vitamins</td>
			<td style="width:127px;">Duchefa Biochemia</td>
			<td style="width:119px;">M0222.0050</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">RNase free surface decontaminant</td>
			<td style="width:127px;">Apex</td>
			<td style="width:119px;">10-228</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Diethyl pyrocarbonate (DEPC)</td>
			<td style="width:127px;">Bio Basic Inc.&#160;</td>
			<td style="width:119px;">D801154</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Sodium chloride (NaCl)</td>
			<td style="width:127px;">MDBio, Inc.</td>
			<td style="width:119px;">101-7647-14-5</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:321px;">Potassium chloride, crystal (KCl)</td>
			<td style="width:127px;">J.T. Baker</td>
			<td style="width:119px;">7447-40-7</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:321px;">Sodium phosphate dibasic (NA2HPO4)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">7558-79-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:321px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">7778-77-0</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Paraformaldehyde (PFA)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">16005</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Sodium hydroxide (NaOH)</td>
			<td style="width:127px;">Showa</td>
			<td style="width:119px;">19430160</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:321px;">Sulfuric acid (H2SO4)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">7664-93-9</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Glutaraldehyde solution</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">111-30-8</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Polyoxyethylene 20 sorbitan monolaurate (Tween 20)</td>
			<td style="width:127px;">J.T. Baker</td>
			<td style="width:119px;">9005-64-5</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Octyl Phenol Ethoxylate (Triton X100)</td>
			<td style="width:127px;">J.T. Baker</td>
			<td style="width:119px;">9002-93-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Glass scintillation vials</td>
			<td style="width:127px;">Newastar</td>
			<td style="width:119px;">DG60805-00020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Desiccator vacuum</td>
			<td style="width:127px;">Violet Bioscience Inc.</td>
			<td style="width:119px;">TS-402030</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Rotary vane pump RZ2.5</td>
			<td style="width:127px;">Vacuubrand</td>
			<td style="width:119px;">36149406</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Ethanol</td>
			<td style="width:127px;">J.T. Baker</td>
			<td style="width:119px;">64-17-5</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Sub-X&#160;</td>
			<td style="width:127px;">Leica Surgipath</td>
			<td style="width:119px;">3803670</td>
			<td style="width:167px;">Xylene substitute</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Paraplast Plus&#160;</td>
			<td style="width:127px;">Leica Surgipath</td>
			<td style="width:119px;">39602004</td>
			<td style="width:167px;">Tissue embedding paraffin</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">SUPERFROST micro slide glass</td>
			<td style="width:127px;">Matsunami</td>
			<td style="width:119px;">S7441</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:321px;">FSC 22 Clear&#160;</td>
			<td style="width:127px;">Leica Surgipath</td>
			<td style="width:119px;">3801480</td>
			<td style="width:167px;">Optimal Cutting Temperature compound (OCT)&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Xylenes, Purified</td>
			<td style="width:127px;">Leica Surgipath</td>
			<td style="width:119px;">3803665</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Hematoxylin</td>
			<td style="width:127px;">Merck</td>
			<td style="width:119px;">HX305311</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Hydrochloric acid (HCl)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">7647-01-0</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">Micromount</td>
			<td style="width:127px;">Leica Surgipath</td>
			<td style="width:119px;">3801731</td>
			<td style="width:167px;">Mounting medium</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">pGEM T-easy vector</td>
			<td style="width:127px;">Promega</td>
			<td style="width:119px;">A1360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Proteinase K</td>
			<td style="width:127px;">Roche</td>
			<td style="width:119px;">3115879001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">SP6/T7 DIG RNA Labeling Kit</td>
			<td style="width:127px;">Roche</td>
			<td style="width:119px;">11175025910</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:321px;">NBT/BCIP stock solution</td>
			<td style="width:127px;">Roche</td>
			<td style="width:119px;">11681451001</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

hybrid-cut: an improved sectioning method for recalcitrant plant tissue samples

الحفاظ على قسم النبات سلامة الضروري لدراسة الهياكل التشريحية مفصلة في الخلوية والأنسجة، أو حتى على مستوى الأعضاء. ومع ذلك، فإن بعض الخلايا النباتية لها جدران الخلايا صلبة، الألياف صعبة وبلورات (أكسالات الكالسيوم، والسليكا، وما إلى ذلك)، ونسبة عالية من المياه التي غالبا ما يعطل سلامة الأنسجة خلال باجتزاء الأنسجة النباتية. وتحدد هذه الدراسة الأنسجة قطع الهجين طريقة بسيطة باجتزاء. هذا البروتوكول بتعديل أسلوب باجتزاء القائم على البرافين ويحسن سلامة أقسام الأنسجة من النباتات المختلفة. كانت جزءا لا يتجزأ من الأنسجة النباتية في البارافين قبل باجتزاء في ناظم البرد في -16 درجة مئوية. باجتزاء تحت درجة حرارة منخفضة تصلب كتل البارافين، وانخفاض تمزيق والخدش، وتحسين سلامة الأنسجة بشكل كبير. تم تطبيق هذا البروتوكول بنجاح إلى الكالسيوم الأنسجة فالاينوبسيس السحلبية-أكسالات الغنية وكذلك الأنسجة المتمردة مثل ORGA الإنجابنانوثانية وأوراق الأرز، والذرة، والقمح. وبالإضافة إلى ذلك، ونوعية عالية من أقسام الأنسجة من الهجين قص يمكن استخدامها في تركيبة مع التهجين في الموقع (ISH) لتوفير أنماط التعبير المكانية للجينات الفائدة. وفي الختام، وهذا البروتوكول هو مفيد بشكل خاص للالأنسجة النباتية المتمردة التي تحتوي على الكريستال عالية أو محتوى السيليكا. تمكن أقسام الأنسجة ذات نوعية جيدة الدراسات البيولوجية المورفولوجية وغيرها.

Watch this Scientific Journal Video about Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples at JoVE.com

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

الهجين قطع: طريقة محسنة باجتزاء لعينات الأنسجة النباتية المتمردة

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

hybrid-cut-an-improved-sectioning-method-for-recalcitrant-plant

Nanyang Technological University

Research

JoVE Journal

Biology

18.8K Views.  Academia Sinica. This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Video: Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Sectioning رقيقة من الاستعدادات لشريحة المناعية

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

اختبار تسامح النيكوتين في المن باستخدام تجربة حمية الاصطناعي

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

آغار بلوك عوالم مصغرة لتحليل الأنسجة النباتية الخاضعة للرقابة من قبل الفطريات الهوائية

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

التنميط الجيني للعينات النباتية والحيوانية دون تنقية DNA قبل

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

لتحسين طريقة لقياس دقة وسرعة الأداء في الطيران<em&gt; ذبابة الفاكهة</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

وسيلة فعالة للالكمي، التحليل خلية واحدة من لونين تعديل والعمارة النووية في جبل لالجامع بويضات في<em&gt; نبات الأرابيدوبسيس</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

طريقة لقياس آخر من Deubiquitinating الانزيمات في خطوط الخلايا وعينات الأنسجة

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

سريع الهواء الجاف اسقاط كروموسوم طريقة التحضير مناسبة لFISH في النباتات

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

تحسين تقنيات السويسري المتداول لإعداد المعوية الأنسجة لالمناعى وتحليلات مناعي

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

طريقة سهلة لمحطة Polysome التنميط

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...