نشكر غراهام بول لخدمة كرجل الكاميرا في عرض الفيديو.

الموت جوعا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أولا ، تجويع الطيران. </li><li style=";text-align:right;direction:rtl"> وضع قطعة صغيرة من كيم يمسح بماء غارقة في القنينة الفارغة. </li><li style=";text-align:right;direction:rtl"> نقل 20-30 جيدا الذباب يتغذى على القارورة الفارغة. </li><li style=";text-align:right;direction:rtl"> تحقق كل 12 ساعة للتأكد من -24 ورقة رطبة. </li><li style=";text-align:right;direction:rtl"> تجويع الطيران ل36-72 ساعة. </li></ol> الإصلاح الذبابة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تأخذ واحدة تحلق في الشافطة. </li><li style=";text-align:right;direction:rtl"> مكان غيض الصفراء 200 ميكرولتر خلال الافتتاح. التقليب في وقت واحد وتهب على ذلك ، يمكنك ضغط على الطيران تصل الى الحافة الصفراء. </li><li style=";text-align:right;direction:rtl"> بلطف تطبيق بعض الهواء لدفع يطير الى نقطة حيث انها لا تستطيع التحرك. </li><li style=";text-align:right;direction:rtl"> باستخدام أسلاك شائكة ، ووضع علامة على ما يقرب من عين الذبابة ، تمتص ثم يعود وقطعوا رأس. </li><li style=";text-align:right;direction:rtl"> ضربة بلطف يعود الى الموقف ، ولكن قبل الوصول إلى النهاية ، وجعل قطع الميل في موقف حيث خرطوم سوف يكون موجودا. يمكنك أن تفعل هذا دون قطع الميل ، لكنها بالتأكيد من شأنه أن يجعل العملية التالية أسهل. </li></ol> تحضير الفتائل السكروز <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لف الشريط 6 ملم واسعة من Kimwipes ® رقة في الموضوع ، ومزق إربا صغيرة من مخروط ، مثل الفتيل شكل (&lt;1 سم). </li><li style=";text-align:right;direction:rtl"> وقد انخفضت هذه الفتائل الصغيرة إلى حلول السكروز أو المياه وعرضها على خرطوم بجعل الاتصال. </li></ol> إعداد الضوابط وحيوانات التجارب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل اختبار أي tastants ، واعطاء الحل السكروز 4 ٪ من الطيران. هذا بمثابة تحكم إيجابية. تجاهل أي أن يطير لم تستجب. </li><li style=";text-align:right;direction:rtl"> المقبل ، واختبار درهم 2 0 على الطاير. هذا بمثابة سيطرة سلبية. تجاهل يطير اذا لم تستجيب. </li><li style=";text-align:right;direction:rtl"> اختبار هذه الضوابط بعد اختبار tastants الخاص. </li></ol>

<ol>
	<li>Dethier, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Hungry Fly. , (1976).</li></ol>

في هذا العرض ، لقد مررنا جميع الخطوات لتمديد خرطوم مقايسة الاستجابة. الأسلوب أصوات جميلة واضحة ولكن كما هو الحال في العديد من المقايسات السلوك الأخرى ، والعديد من الأشياء الصغيرة يمكن مقايسة منع من العمل. مررنا مثل هذه الخطوات الكثيرة التي تحتاج إلى عناية دقيقة. هنا ، أود التأكي...

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proboscis extension response (per) assay in drosophila

خرطوم ردا التمديد (PER) هو فحص سلوك الذوق الذي تم استخدامه في الذباب ، وكذلك في نحل العسل. على سطح الفم الذبابة (labellum) ، وهناك الشعر هياكل شبيهة تدعى الخلايا العصبية التي تضم sensilla الذوق. عندما مادة جذابة يجعل الاتصال على labellum ، ذبابة خرطوم يمتد لتستهلك المواد. خرطوم ملحق مواجهتها (PER) مقايسة التدابير سلوك هذا الرد الذوق ، وأنه هو وسيلة مفيدة لمعرفة أذواقهم الغذائية في ذبابة واحدة. حلول من السكريات المختلفة ، مثل الجلوكوز والسكروز والفركتوز ، وجذابة للغاية للطيران. يمكن أيضا أن تأثير المواد مكره يتم اختبارها مع تخفيض PER عندما مختلطة في حل الحلو. على الرغم من بساطة الإجراء الأساسي ، وهناك الكثير من الامور التي يمكن منعها من العمل. واحدة من العوامل التي تتطلب اهتماما هو الدولة الذبابة استجابة. الوقت التجويع اللازمة لجعل السفر إلى دولة متجاوبة السليم يختلف جذريا 36-72 ساعة. أقمنا مجموعة من الضوابط لتقييم حالة الذبابة والفرز الذي يسمح للخروج من الحيوانات فرادى أو غير متجاوبة فرط استجابة. عامل آخر مهم هو مستوى التأثير والموقف من الاتصال إلى labellum ، التي سيكون من الصعب وصف بالكلمات. يوضح هذا العرض الفيديو كل هذه جنبا إلى جنب مع العديد من التحسينات الأخرى التي من شأنها أن تزيد من استنساخ هذا الأسلوب.

Watch this Scientific Journal Video about Proboscis Extension Response PER Assay in Drosophila at JoVE.com

Proboscis Extension Response PER Assay in Drosophila

خرطوم ردا تمديد أو PER هو مقايسة سلوك الذوق الذي تم استخدامه في الذباب ، وكذلك في نحل العسل. عندما خرطوم يجعل الاتصال مع مادة جذابة ، ذبابة خرطوم يمتد لاستهلاك هذه المادة. حلول مختلفة من السكريات هي جذابة جدا للطيران.

خرطوم ملحق مواجهتها (PER) الفحص في ذبابة الفاكهة

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

proboscis-extension-response-per-assay-in-drosophila

Research

JoVE Journal

Biology

Proboscis Extension Response (PER) Assay in Drosophila

30.2K Views.  Yale University. خرطوم ردا تمديد أو PER هو مقايسة سلوك الذوق الذي تم استخدامه في الذباب ، وكذلك في نحل العسل. عندما خرطوم يجعل الاتصال مع مادة جذابة ، ذبابة خرطوم يمتد لاستهلاك هذه المادة. حلول مختلفة من السكريات هي جذابة جدا للطيران.

Video: Proboscis Extension Response PER Assay in Drosophila

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

التمهيدي لقطة فرعي : استرجاع المستهدفة تسلسل الحمض النووي من مصادر المتدهورة بالديون

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

الكمي بؤر γH2AX في الاستجابة للإشعاع المؤين

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

التصوير تغيير الشكل في الخلية الحية ذبابة الفاكهة الأجنة

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

المظهري التحليل وعزل الخلايا الجذعية المكونة للدم الفئران والأسلاف النسب، الملتزم

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

يعيش التصوير من تخليص الخلية أفكارك أثناء<em&gt; ذبابة الفاكهة</em&gt; مرحلة التطور الجنيني

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

عزل الكائنات الدقيقة في التربة الأصلية مع المحتملة لتحطيم القابلة للتحلل أفلام الجرثومة البلاستيكية المستخدمة في الزراعة

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

قياس الاستجابة التغذية الناجم عن الجلوتاثيون في حيدرة

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

استخدام البروتينات النووية الذاتية من الخلايا المزروعة تحت ظروف التاكسج بالانزيم Co-إيمونوبريسيبيتيشن

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

الفحص الضوئي القائم علي الضوء لتحليل دروتواكسيس

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

تشريح وإصلاح وتصور ذبابة الفاكهة العذراء Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...