فإن الكتاب أود أن أشكر Ikiel Ptito لدعمه الفني المستمر. NSERC منحة لعضوية البرلمان.

الجزء 1 : قبل معالجة الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وينبغي أن الأنسجة perfused جيدا مع بارافورمالدهيد ، غلوتارالدهيد ، أو الفورمالين. ويمكن تحقيق ذلك من خلال نضح transcardial القياسية المستخدمة عادة لحصاد الأجهزة الأخرى. في هذه الدراسة كان مخدرا بعمق هذا الموضوع مع هيدروكلوريد الكيتامين (10 ملغم / كغم ، IM) ، الموت الرحيم مع جرعة زائدة من (الرابع 25 ملغ / كلغ ،) والصوديوم بنتوباربيتال perfused transcardially مع PBS حتى 0،1 م ويتبع exsanguinated.This تماما بواسطة حلا بارافورمالدهيد 4 ٪ في برنامج تلفزيوني لمدة 5 دقائق (~ 1 ليتر). </li></ol> الجزء 2 : حظر التجسيمي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل أن يضع رأسه في الإطار التجسيمي إحداثيات صفر الطائرة Horsley-Clarke/interaural يتعين اتخاذها. هذا هو نقطة الوسط بين النظرية والأذنين. لقياس هذه الطائرة ، وأشرطة الأذن الحاجة إلى تركيبها على قدم المساواة في الجهاز ، ثم ضع شفرة المشرط في الذراع تتلاعب التجسيمي وقياس نقطة الوسط بين القضبان الأذن. وهذا أمر مهم لتحديد مكان كتلة النسيج على أساس الاحتياجات البحثية. بمجرد الانتهاء من هذه الجمجمة يحتاج إلى أن تكون مستعدة لتثبيت الجهاز في التجسيمي. </li><li style=";text-align:right;direction:rtl"> من أجل وضع الرأس في الإطار التجسيمي والفك السفلي يجب أن تكون إزالة مع rongeurs العظام ومشرط. بالإضافة إلى ذلك ينبغي أن تتم إزالة الجلد والعضلات والنسيج الضام ، من أجل كشف الجمجمة. حالما يتم إزالة النسيج الضام من الجمجمة ، وتعريض الدماغ عن طريق التقطيع بعيدا calvaria (الجزء جدولي العظم القذالي الجداري ، وكذلك في العظام الأمامية الإعلانية). في الجزء التجربة الحالية لديه كل استعداد calvaria أزيلت. يجب الحرص على عدم إزالة العظام الزمنية ، لأن قنوات الأذن سوف تحتاج إلى أن تكون سليمة لوضع الرأس في الإطار التجسيمي. يجب إزالة هذه المسألة الجافية المتبقية من الدماغ عرضة للخطر. الجمجمة هو الآن على استعداد لوضعها في الإطار التجسيمي. </li><li style=";text-align:right;direction:rtl"> بنفس الطريقة التي يمكن للمرء أن إجراء جراحة التجسيمي ، وضبط العين والحنك والأذن مثل هذه الحانات التي يتم إصلاحها بشكل آمن الرأس في جهاز التجسيمي. ضع التجسيمي تتلاعب في إحداثيات محددة سلفا الأمامي / الخلفي (A / P) ونقل إلى الجانب تتلاعب الوحشي للدماغ. انخفاض ببطء النصل في الدماغ ، ورفع النصل تماما من الدماغ ، ثم ينتقل بشكل إعلامي وانخفاض النصل مرة أخرى. كرر هاتين الخطوتين حتى نصل وصل الجانب الوحشي من نصف الكرة المعاكس. هذا يكمل اللبنة الأولى الاكليلية. للكتل الإكليلية اللاحقة نقل 1cm تتلاعب في المحور A / P ، وكرر حتى تم حجب الدماغ كله. </li></ol> الجزء 3 : إزالة الدماغ من الجمجمة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة الرأس من الإطار التجسيمي والاستمرار في الدماغ يتعرض في كف يدك. محاولة لتأمين الدماغ عن طريق وضع تدليل طفيفة في الدماغ في راحة يدك ومكانك الاصبع الخنصر في الفص الجبهي ، وهذا يقلل من حركة الدماغ في الجمجمة. من أجل منع جفاف سطح حنوني من الدماغ ، ضع قطعة من الشاش المبلل في برنامج تلفزيوني عبر الدماغ. عقد رئيس بحزم إلى جانب جمجمة وعظام رقاقة بعيدا المتبقية القذالي الصدغي وجنبا إلى جنب مع العمود الفقري. هذا الكشف عن الجوانب الأساسية والجانبية من الدماغ. أخيرا إزالة أي العظام المتبقية والعظم الجبهي الأنفي ، مما سيتيح الوصول إلى بصيلات الشم. ومن المهم لإزالة العظم الجبهي الماضي بسبب ما قمت بإزالة قاعدة الجمجمة والدماغ يتحرك قليلا ويمكن أن تصبح تلف الفص الجبهي على حواف خشنة من العظام fontal. قطع وإزالة ما تبقى المسألة الجافية. رفع بعناية الجزء الأمامي من الدماغ ، وانزلاق تحت مشرط الدماغ وخالية من الدماغ في الجمجمة. </li></ol> الجزء 4 : قياسات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> هناك عدد من القياسات المفيدة التي يمكن إجراؤها قبل التجميد. على سبيل المثال ، محور P / من الدماغ مع الفرجار. وعلاوة على ذلك يمكن أن تقاس كثافة محددة من الدماغ وزنها ، وقياس حجم عن تشريد الماء في الاسطوانة ثم تقسيم الوزن عن طريق حجم النزوح (الجدول 1). </li></ol> الجزء 5 : إنهاء حجب الدماغ. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عادة عندما يتم إدراج شفرة المشرط في الدماغ ليست طويلة بما يكفي لاختراق تماما الكامل الظهري البطني مدى الدماغ. بمجرد إزالة المخ ويتم الحصول على قياسات الإجمالي ، واتخاذ تشريح الأنسجة النصل وإنهاء حظر الدماغ من خلال إلى الجانب البطني من الدماغ (الشكل 2). </li><li style=";text-align:right;direction:rtl"> قبل تجميد الكتل ، فمن الضروري cryoprotect النسيج في حلول السكروز متدرج PBS - مخزنة (10 ، 20 ، و 30 ٪) في المحافظة 4 ˚ C حتى البواليع الدماغ. يستغرق عادة ما بين عشية وضحاها في الحضانة 10 ٪ ، 2-3 أيام في 20 ٪ و 3-5 أيام إضافية في 30 ٪ للبنات لتنزل الى قاع الإناء. تغيير يومي الخامسويوصى ه حل السكروز 30 ٪. </li></ol> الجزء 6 : النتائج الممثل هناك عدد من القياسات المورفولوجية الجسيمة التي يمكن إجراؤها مرة واحدة تمت إزالة الدماغ من الجمجمة. وتشمل هذه بطول / P ، والوزن ، والكثافة المحددة (الجدول 1). نحن كتلة عموما في الدماغ 6-7 قياس كتل 1cm (الشكل 1). وصورت كل قطعة ثم (الشكل 2) ، ويمكن كذلك اعتمادا على تشريح الاحتياجات البحثية أو المعدة للتجميد في حلول السكروز متدرج. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> موضوع </td><td> طول / P (ملم) </td><td> الوزن (جرام) </td><td> المياه بالتشرد (مل) </td><td> الكثافة المحددة (ز / مل) </td></tr><tr><td> O2303 - 2 - 1 - 1 </td><td> 64.3 </td><td> 28.1 </td><td> 24 </td><td> 1،171 </td></tr><tr><td> O5180 - 1 </td><td> 71.3 </td><td> 38.7 </td><td> 34 </td><td> 1،138 </td></tr><tr><td> O2708 - 3 - 1 </td><td> 62.8 </td><td> 28.7 </td><td> 26 </td><td> 1،104 </td></tr><tr><td> O9184 - 4 - 2 </td><td> 65.3 </td><td> 29.5 </td><td> 24 </td><td> 1،229 </td></tr><tr><td> N459 - 1 - 14-2 </td><td> 68.2 </td><td> 31.6 </td><td> 26 </td><td> 1،215 </td></tr><tr><td> AVERAGE </td><td> 66.38 </td><td> 31.32 </td><td> 26.8 </td><td> 1،171 </td></tr><tr><td> STD DEV </td><td> 3.38 </td><td> 4.33 </td><td> 4.17 </td><td> 0،052 </td></tr></tbody></table> الجدول 1. القياسات المورفولوجية الإجمالي في نصف الكرة الأرضية حق Vervets شهر 5 قديم <img src="/files/ftp_upload/1259/1259fig1.jpg" alt="الشكل 1" /> <img src="/files/ftp_upload/1259/1259fig2.jpg" alt="الشكل 1.5" /> الشكل 1. الخطط للطائرات تستخدم لحجب الاكليلية الدماغ ، وهذا مثال على الدماغ الكبار الفرفت تخريجها. مثال على هذا الإجراء حظر. متباعدة خطوط عمودية هنا في 1cm ، وتنتج عادة من 7 كتل الإكليلية كل نصف الكرة الغربي. <img src="/files/ftp_upload/1259/1259fig3.jpg" alt="الشكل 2" /> الشكل 2. كتل من نسيج المخ في الفضاء التجسيمي. سوف تسفر كل كتلة ما يقرب من 200 المقاطع التي اتخذت في 50μm. مع هذا البرنامج سوف تؤخذ عينات أكثر من 1200 من خلال المقاطع القشرة و400-500 إضافية من المخيخ عند شرائح في الطائرة الاكليلية.

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The authors have nothing to disclose.

سانت كيتس والفرفت (Chlorocebus aethiops sabeus) هو الرئيسيات العالم القديم مع أنماط مماثلة ، ومعدلات نمو الدماغ القشرية وتحت القشرية إلى أن من البشر. وقد استخدم هذا النوع لنموذج معقدة اضطرابات سلوكية مثل السلوك البشري القلق ، ارتفاع ضغط الدم 8 ، 9 استئصال نصف الكر?...

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<td>Scalpel</td>
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<td>Fine Science Tools</td>
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<td>Scalpel blades</td>
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<td>Fine Science Tools</td>
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<td>Any surgical scissors are sufficient</td>
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<td>Rongeurs</td>
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<td>Fine Science Tools</td>
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<td>Filter paper</td>
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<td>Fisher</td>
<td>09-924-150</td>
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dissecting the non-human primate brain in stereotaxic space

استخدام الرئيسيات غير البشرية ، نموذجا متعدية لفهمنا لعمليات النمو والشيخوخة في البشر<sup&gt; 1-6</sup&gt;. بالإضافة إلى ذلك ، قد أتاحت استخدام الرئيسيات غير البشرية مؤخرا الفرصة لنموذج طبيعي اضطرابات نفسية معقدة ، مثل تعاطي الكحول<sup&gt; 7</sup&gt;. نحن هنا وصف تقنية لحجب الدماغ في الطائرة الاكليلية من قرد الفرفت (<em&gt; Chlorocebus aethiops sabeus</em&gt;) في الجمجمة سليمة في الفضاء التجسيمي. وصف الأسلوب يوفر هنا طائرة مستوى القسم بين الكتل والموضوعات والفروع الجزئية يقلل بين الكتل. Sectioning كتلة من الأنسجة في الطائرة الاكليلية يسهل أيضا ترسيم مساحة الفائدة. هذا الأسلوب يوفر كتل التحكم بحجم نصف الكرة الأرضية منذ واحد من قرد الفرفت الغلة أكثر من 1200 المقاطع عند تشريح في 50μm. وعلاوة على ذلك عن طريق منع الدماغ إلى كتل 1cm ، تسهل تغلغل السكروز لcyroprotection ويسمح شرائح كتلة على ناظم البرد العادية.

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Dissecting the Non-human Primate Brain in Stereotaxic Space

الرئيسيات غير البشرية هي الأنواع الهامة متعدية لفهمنا لمعالجة العادي من الدماغ. يمكن للمنظمة التشريحية للدماغ الرئيسيات توفير معلومات هامة في الظروف العادية والمرضية لدى البشر.

تشريح الدماغ البشري غير الرئيسيات في الفضاء التجسيمي

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

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10.0K Views.  University of Montreal. الرئيسيات غير البشرية هي الأنواع الهامة متعدية لفهمنا لمعالجة العادي من الدماغ. يمكن للمنظمة التشريحية للدماغ الرئيسيات توفير معلومات هامة في الظروف العادية والمرضية لدى البشر.

Video: Dissecting the Non-human Primate Brain in Stereotaxic Space

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

وحيد الخلية في الجسم الحي Electroporation داخل الدماغ سليمة النامية

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

التجسيمي جراحة للبقاء في القوارض

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

تشريح والتسجيل من مفرق العضلية جيم ايليجانس

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

عبارة في الدماغ : تشريح العين الرئيسيات

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2, generates reliable structural data for biological features in the mammalian brain3. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

معرفة ما يهم : التجسيم غير متحيز في الدماغ البشري غير الرئيسيات

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4.
Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling.
The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

مسار مشي لبيانات التحاليل دراسة النشاط الفضائي في الوقت

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

أخذ عينات الدم من الوريد الذيل الأفقي من الفئران

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

ميكروبونكتوري الفضاء بومان في الفئران التي يسرتها 2 فوتون مجهرية

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

أديبو--واضحة: منديل مسح طريقة لتصوير ثلاثي الأبعاد للأنسجة الدهنية

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

عكس التشريح والبيانات ديسيكت تكشف خلاف ذلك خفية في تطور الرئيسيات الوجه

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...