وأود أن أشكر الدكتور جنيفر كيرتس للصور في الشكل. (1) وجيريمي والدكتور فلورنسا فاغان مارلو لقراءة نقدية للمخطوطة. ويدعم عملنا المعاهد الوطنية للصحة منح 1R01GM088202.

<ol>
	<li>Harvey, K. F., Pfleger, C. M., Hariharan, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+Mst+ortholog,+hippo,+restricts+growth+and+cell+proliferation+and+promotes+apoptosis.">The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis.</a> Cell. 114, 457-457 (2003).</li><li>Secombe, J., Li, L., Carlos, L., Eisenman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Trithorax+group+protein+Lid+is+a+trimethyl+histone+H3K4+demethylase+required+for+dMyc-induced+cell+growth.">The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth.</a> Genes Dev. 21, 537-537 (2007).</li><li>Cagan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Drosophila+eye+differentiation.">Principles of Drosophila eye differentiation.</a> Curr Top Dev Biol. 89, 115-115 (2009).</li><li>Jenny, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+cell+polarity+signaling+in+the+Drosophila+eye.">Planar cell polarity signaling in the Drosophila eye.</a> Curr Top Dev Biol. 93, 189-189 (2010).</li><li>Tomlinson, A., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+in+the+Drosophila+Ommatidium.">Cell fate in the Drosophila Ommatidium.</a> Dev. Biol. 123, 264-264 (1987).</li><li>Tomlinson, A., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+differentiation+in+the+Drosophila+ommatidium.">Neuronal differentiation in the Drosophila ommatidium.</a> Dev. Biol. 120, 366-366 (1987).</li><li>Jenny, A., Darken, R. S., Wilson, P. A., Mlodzik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prickle+and+Strabismus+form+a+functional+complex+to+generate+a+correct+axis+during+planar+cell+polarity+signaling.">Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling.</a> Embo J. 22, 4409-4409 (2003).</li><li>Zheng, L., Zhang, J., Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=frizzled+regulates+mirror-symmetric+pattern+formation+in+the+Drosophila+eye.">frizzled regulates mirror-symmetric pattern formation in the Drosophila eye.</a> Development. 121, 3045-3045 (1995).</li><li>Reinke, R., Zipursky, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+interaction+in+the+Drosophila+Retina:+The+bride+of+sevenless+Gene+is+required+in+photoreceptor+cell+R8+for+R7+cell+development.">Cell-cell interaction in the Drosophila Retina: The bride of sevenless Gene is required in photoreceptor cell R8 for R7 cell development.</a> Cell. 55, 321-321 (1988).</li><li>Wolff, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=strabismus,+a+novel+gene+that+regulates+tissue+polarity+and+cell+fate+decisions+in+Drosophila.">strabismus, a novel gene that regulates tissue polarity and cell fate decisions in Drosophila.</a> Development. 125, 1149-1149 (1998).</li><li>Winter, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Rho-associated+kinase+(Drok)+links+Frizzled-mediated+planar+cell+polarity+signaling+to+the+actin+cytoskeleton.">Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton.</a> Cell. 105, 81-81 (2001).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117, 1223-1223 (1993).</li><li>Stocker, H., Gallant, P., Dahmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods Mol Biol. 420, 27-27 (2008).</li></ol>

الإعلان عن أي تضارب في المصالح.

ذبابة الفاكهة واستخدام نموذج حي ، وشاشات وراثية أدت إلى تحديد العديد من الأعضاء المؤسسين للعائلات الجينات الأساسية لمعظم المسارات المحفوظة للغاية مما يشير الى ارتفاع حقيقيات النواة بما فيها البشر. منذ ذلك الحين ، تحت ظروف المختبر ، والعين هي وظيفية يمكن الاستغناء عنها من أجل البقاء ، والعين هي الأنسجة وخاصة مناسبة تماما لاكتشاف وظائف الجينات رواية وتقييم الشبكات الجينية. تحليل التركيبية للعين الذبابة باستخدام الطريقة الموصوفة وبالتالي أدى إلى اكتشافات الأساسية ذات الصلة من أجل التنمية والمرض. في البداية ، تم إجراء تحليل وحيد الخلية نسيلي باستخدام الأشعة السينية التي يسببها استنساخ جنبا إلى جنب مع علامات الخلية يعرف الحكم الذاتي مرتبطة ارتباطا وثيقا المتنحية. في الآونة الأخيرة ، وقد سهلت توافر نظام FLP / FRT لتوليد الحيوانات المستنسخة كثيرا من التحليل المظهرية للطفرات المميتة في أقسام العيون 6،9. ولا يقتصر تحليل المقاطع إلى مقاطع عرضية العين الموصوفة هنا. إذا رغبت في ذلك ، يمكن محاذاة الرؤوس في أي اتجاه في قوالب ويمكن الحصول على المقاطع العرضية لدراسة الطبقات الأعمق في رأسه مثل الصفيحة والنخاع. بروتوكول الموصوفة هنا هو بالتالي وسيلة لتحليل تنوعا من البالغين العين ورأس ذبابة الفاكهة الهياكل.

معدات عامة : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لتربية ذبابة عامة انظر 10 </li><li style=";text-align:right;direction:rtl"> مجهر تشريح القياسية (مثل نيكون ستيريو تكبير المجهر SMZ - 645) ، ويفضل مع عصابة مصدر للضوء لمنع يلقي ظلالا من يديك. </li><li style=";text-align:right;direction:rtl"> CO 2 وسادة يطير ، مع تغطية ورقة Whatman 3mm ل توفير حماية ضد الأوساخ أثناء تشريح. </li><li style=";text-align:right;direction:rtl"> الشرائح المغلفة الجيلاتين : الجيلاتين ويذوب 10G 1G CrKSO 4 × 12 H 2 O في المياه 1L على طبق من ذهب للتدفئة (تقريبا جلب ليغلي ، ويأخذ ما يقرب من 2 ساعة). تراجع الشرائح غسلها جيدا (الصابون) وتشطف (DH 2 O) في حملة الى حل الجيلاتين والهواء الجاف المغطاة برقائق بين عشية وضحاها. </li><li style=";text-align:right;direction:rtl"> فرن التسخين (70 درجة مئوية ، ومناسبة تماما فراغ الأفران القديمة التي كانت تستخدم لتجفيف مرشح النيتروسليلوز لهذا الغرض). </li><li style=";text-align:right;direction:rtl"> مشراح قادرة على sectioning سامسونج (0.5-1 أبواب سميكة ميكرومتر) ، مثل تلك المستخدمة من قبل مرافق المجهر الإلكتروني. نستخدم MT5000 Sorvall مشراح مستدق مع الماس Histo سكين الجودة. السكاكين والزجاج العمل أيضا. </li></ul> تثبيت الحلول : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يعد حل الأسهم من 0.2 م العازلة الفوسفات الصوديوم ، ودرجة الحموضة 7.2. </li><li style=";text-align:right;direction:rtl"> إعداد غلوتارالدهيد الصوديوم 2 ٪ في 0.1M العازلة الفوسفات ودرجة الحموضة 7.2 (يمكن تخزينها في 4 أسابيع 4 درجات مئوية ل). جعل الأقل بما يكفي ليكون في التركيب الوراثي 200μl تريد تضمين. تبقي على الجليد. </li><li style=";text-align:right;direction:rtl"> الأوزميوم الحل : 2 ٪ 4 في أوسو الصوديوم 0.1M العازلة الفوسفات ودرجة الحموضة 7.2. تقديم ما يكفي فقط أن يكون في التركيب الوراثي 200μl تريد تضمين ، لأن لا يمكن أن يكون الحل المخزنة. تبقي على الجليد. </li></ul> غلوتارالدهيد وأوسو 4 سامة للغاية وينبغي التعامل معها بعناية فائقة في غطاء جيد التهوية. نصائح استخدام التصفية عند التعامل مع أوسو 4 لمنع اسوداد من ماصة الخاص. الجفاف : جعل 30 ٪ ، 50 ٪ ، 70 ٪ ، (80 ٪) ، 90 ٪ ، والحلول الإيثانول بنسبة 100 ٪. بارد ويفضل 30 ٪ و 50 ٪ و 70 ٪ من الإيثانول على الجليد قبل الاستخدام. تلطيخ الحل : 1 ٪ طولويدين الأزرق في البوراكس 1 ٪. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th></th><th> ناعم </th><th> الثابت </th></tr><tr><td> والراتنج </td><td> 54G </td><td> 50G </td></tr><tr><td> تصليب B </td><td> 44.5g </td><td> 50G </td></tr><tr><td> مسرع C </td><td> 2.5G </td><td> 1.75g </td></tr><tr><td> الملدنات D </td><td> 10G </td><td> 0.75g </td></tr></tbody></table> الجدول 1. الراتنج إعداد لإعداد الراتنج ، وبعناية ، ولكن خلط جميع المكونات جيدا في كوب من البلاستيك باستخدام مغناطيسي مع بقضيب كبير. تجنب الفقاعات. بعد قسامة ، والخلط في قوارير أو حاويات قياسية التلألؤ وتجميد مماثل على -20 درجة مئوية. استخدام الراتنج ميسرة لجميع الطلبات ما لم يكن لديك EM مرفق يسألك عن صعوبة الصياغة. استخدام قفازات للتعامل مع الراتنج (مادة مسرطنة في شكله unpolymerized). لتتبلمر راتنج على المعدات والنفايات ، ويخبز في 70 درجة مئوية خلال الليل. ثم قد يكون التخلص منها بأمان من النفايات والمعدات تنظيفها وإعادة استخدامها. ويمكن تنظيف تسرب راتنج unpolymerized مع الأيزوبروبانول. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> الكاشف </th><th> المزود </th><th> فهرس العدد </th></tr><tr><td> يطير سادة </td><td> على سبيل المثال جينيسي </td><td> 59-119 </td></tr><tr><td> غلوتارالدهيد </td><td> سيغما </td><td> G7526 - 10 X 10 ML </td></tr><tr><td> 4 OSO </td><td> Polysciences ، وشركة </td><td> 0972A - 20 </td></tr><tr><td> Propyleneoxide </td><td> الصياد </td><td> 04332-1 </td></tr><tr><td> مشرط المقابض ، ل# 3 </td><td> الصياد </td><td> 22080046 </td></tr><tr><td> شفرات المشرط # 11 </td><td> الصياد </td><td> 08-916 - 5B </td></tr><tr><td> نقل ماصات </td><td> الصياد </td><td> 13-711 - 7M </td></tr><tr><td> Durcupan (R) ACM الراتنج </td><td> سيغما (Fluka) </td><td> 44610 - 1EA </td></tr><tr><td> إبرة تشريح الصلب </td><td> الصياد </td><td> S17346 </td></tr><tr><td> BEEM العفن تضمين المسطحة </td><td> الإلكترون المجهري الكيمياء. </td><td> 70904-12 </td></tr><tr><td> شفرات الحلاقة المطلية تفلون </td><td> الإلكترون المجهري الكيمياء. </td><td> 71970 </td></tr><tr><td> Q - غيض (مسحات معقمة) </td><td> الصياد </td><td> 14-959-81 </td></tr><tr><td> الشرائح الزجاجية </td><td> الصياد </td><td> 12-550-143 </td></tr><tr><td> تغطية زلات رقم (1) ، 22X60 مم </td><td> الصياد </td><td> 12 - 531K </td></tr><tr><td> الجيلاتين </td><td> الصياد </td><td> ICN96010280 </td></tr><tr><td> الكروم (III) SO 4 K dodecahydrate </td><td> سيغما </td><td> 243361 </td></tr><tr><td> الماس Histo سكين ، 6mm </td><td> Diatome الولايات المتحدة </td><td> صاحب 60 </td></tr><tr><td> طولويدين يا الأزرق </td><td> الصياد </td><td> BP107 - 10 </td></tr><tr><td> البوراكس (بورات ثنائي نا) </td><td> الصياد </td><td> AC20629 - 1000 </td></tr><tr><td> DPX تصاعد المتوسطة </td><td> سيغما </td><td> 44581 - 100ML </td></tr></tbody></table> الجدول 2. المواد

preparation of adult drosophila eyes for thin sectioning and microscopic analysis

منذ فترة طويلة ذبابة الفاكهة تستخدم نظام نموذجي لدراسة التنمية ، ويرجع ذلك أساسا إلى السهولة التي كان لين العريكة وراثيا. على مر السنين ، وقد وضعت مجموعة كبيرة من سلالات متحولة والحيل التقنية للسماح أسئلة معقدة والإجابة عليها في كمية معقولة من الزمن. لقد تم الحصول على الرؤية الاساسية في تفاعل مكونات جميع الممرات الرئيسية المعروفة في إشارة إلى الأمام وعكس الدراسات الجينية ذبابة الفاكهة. وقد ثبت للعين أن يطير جيدا بشكل استثنائي مناسبة لتحليل طفرية ، منذ ذلك الحين ، تحت ظروف المختبر ، والذباب يمكن أن يعيش من دون عيون وظيفية. وعلاوة على ذلك ، يتألف سطح العين الحشرات من بعض العيون 800 وحدة الفرد (أو جوانب ommatidia) الذي شكل منتظم ، وسطح أملس عندما نظرت إلى تشريح تحت المجهر. وهكذا ، فإنه من السهل أن نرى ما إذا كان قد تحور تؤثر على التنمية العين أو النمو من خلال النظر خارجيا لفقدان سطح أملس (&#39;العين الخام&quot; النمط الظاهري ؛ الشكل 1) أو الحجم الكلي العين ، على التوالي (للاطلاع على أمثلة من الشاشات على أساس مورفولوجيا العين الخارجية انظر على سبيل المثال 1). تحليلات مفصلة لاحقة من الظواهر تتطلب تثبيت العين ، تضمين البلاستيك ورقيقة sectioning عيون الكبار. عين ذبابة الفاكهة يطور من القرص العين يسمى تخيلي ، كيس من الخلايا الظهارية التي تتكاثر والتفريق خلال مراحل اليرقات والعذراء (انظر 2 للمراجعة مثلا). كل ommatidium يتكون من 20 زنزانة ، بينهم ثمانية من المستقبلات الضوئية (PR أو R الخلايا ؛ الشكل رقم 2) ، وأربع خلايا مخروطية عدسة إفراز ، الخلايا الصبغية (&#39;السداسي&quot; حول الخلية R - الكتلة) وشعيرات ملف. ومبصرات كل ommatidium ، ومعظم التعرف عليها بسهولة من قبل هذه العضيات الحساسة للضوء ، rhabdomeres ، هي التي نظمت في شبه المنحرف تتكون من &quot;الخارجي&quot; ستة (R1 - 6) واثنين من &quot;الداخلية&quot; المستقبلات الضوئية (R7 / 8 ؛ R8 [التين 2] هو تحت R7 ، وبالتالي ينظر فقط في الفروع من المناطق العميقة من العين). يتم محاذاة بدقة شبه منحرف من كل جانب مع تلك جيرانها والمحاور الشاملة وظهري بطني الأمامي الخلفي من العين (الشكل 3A). على وجه الخصوص ، من ommatidia (الأسهم السوداء والحمراء ، على التوالي) وظهري بطني نصفين من العين هي صور طبق الأصل من بعضها البعض ، وتتوافق مع شكلين مراوان مستو وضعت خلال إشارات الخلية القطبية (انظر على سبيل المثال للمراجعة 3). وصف طريقة لتوليد أقسام العين شبه رقيقة (مثل تلك التي قدمت في الشكل 3) هنا هو تعديل طفيف من وصفها توملينسون في الأصل من قبل وجاهز 4. فهو يسمح للتحليل الصرفي لجميع الخلايا باستثناء الخلايا المخروطية شفافة. بالإضافة ، يمكن استخدام صبغة R - الخلايا (السهام الزرقاء في الشكل 2 و 3) كعلامة خلية مستقلة عن التركيب الوراثي للخلية - R ، ومتطلبات الوراثية وبالتالي من الجينات في مجموعة فرعية من الخلايا يمكن R بسهولة يمكن تحديد 5،6.

Watch this Scientific Journal Video about Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis at JoVE.com

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

نهج موحد لإعداد الكبار<em&gt; ذبابة الفاكهة</emويرد&gt; عيون لشبه رقيقة sectioning والتحليل المجهري الضوء هنا. ويمكن استخدام بروتوكول للتحليل الصرفي من العيوب الجسيمة العين ، أو يمكن استخدامها مع التعديلات وأشار إلى تحديد الاحتياجات من الجينات الوراثية في أنواع معينة من الخلايا في العين (تحليل نسيلي مبصرات مثلا) أو لأغراض التحليل المجهري الإلكترون.

إعداد الكبار ذبابة الفاكهة لعيون Sectioning رقيقة والتحليل المجهري

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

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Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

16.0K Views.  Albert Einstein College of Medicine. نهج موحد لإعداد الكبار ذبابة الفاكهة عيون لشبه رقيقة sectioning والتحليل المجهري الضوء هنا. ويمكن استخدام بروتوكول للتحليل الصرفي من العيوب الجسيمة العين ، أو يمكن استخدامها مع التعديلات وأشار إلى تحديد الاحتياجات من الجينات الوراثية في أنواع معينة من ...

Video: Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

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 ...

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

تسجيلات كاملة من خلية دماغ ذبابة الفاكهة الكبار

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

عالية الدقة تعقب الفيديو للتنقل في الكبار ذبابة الفاكهة السوداء البطن

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

تشريح التلفيف المسنن من الحصين ماوس الكبار

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

إعداد وتطوير الكبار ذبابة الفاكهة الأمخاخ والشبكية للتصوير لايف

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

إعداد ذبابة الفاكهة خلايا S2 الميكروسكوب لايت

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

البارافين ، جزءا لا يتجزأ من والمجمدة أقسام ذبابة الفاكهة</emالعضلات الكبار>

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

ليزر التقاط ميكروديسيكشن من ميشوورك ترابيكولار نقية جداً من عيون الماوس لتحليل التعبير الجيني

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

Automated Charting of the Visual Space of Housefly Compound Eyes

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1&#730;) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

The protocol here describes the measurement of the spatial organization of the visual axes of housefly eyes, mapped by an automatic device, using the pseudopupil phenomenon and the pupil mechanism of the photoreceptor cells.

الرسم الآلي للفضاء البصري للعيون المركبة الذبابة المنزلية

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands ...

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human health. This deficit of chemical safety information is exacerbated by current testing methods in mammals that are expensive, labor-intensive, and time-consuming. Recently, scientists and regulators have been working to develop new approach methodologies (NAMs) for chemical safety testing that are cheaper, more rapid, and reduce animal suffering. One of the key NAMs to emerge is the use of invertebrate organisms as replacements for mammalian models to elucidate conserved chemical modes of action across distantly related species, including humans. To advance these efforts, here, we describe a method that uses the fruit fly, Drosophila melanogaster,&#160;to assess chemical safety. The protocol describes a simple, rapid, and inexpensive procedure to measure the viability and feeding behavior of exposed adult flies. In addition, the protocol can be easily adapted to generate samples for genomic and metabolomic approaches. Overall, the protocol represents an important step forward in establishing Drosophila as a standard model for use in precision toxicology.

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster.

تقييم السمية الكيميائية في ذبابة الفاكهة الميلانوجاستر البالغة

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human ...