ويدعم RVS بواسطة محقق جديد لبدء الأموال من كلية ألبرت اينشتاين للطب في جامعة يشيفا.

<ol>
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ليس لدينا ما يكشف.

وقد وصفت لنا التفاصيل الفنية اللازمة لتلطيخ wholemount ناجحة باستخدام نهج متعدد الاستعمالات المناعى للتعبير عن بروتين كاشفة في الدماغ النامية وتعليم الكبار. باستخدام هذا النهج، يمكن تحليل معقد أنماط التعبير الجزيئية وتضاريس الدماغ تقدير من دون الحاجة إلى شاقة وتستغرق ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> المواد </td><td> وظيفة في بروتوكول </td></tr><tr><td> مضخة نضح (فيشر Scientific/13-876-2) </td><td> يسمح لنضح بما يتفق وبطيئة. </td></tr><tr><td> مقص حاد الطرف (FST/14081-08) </td><td> الاستخدام العام في نضح وتشريح. </td></tr><tr><td> كليل معلومات سرية الملقط (FST/91100-12) </td><td> لتحقيق الاستقرار في قلب لإدخال الإبرة نضح. </td></tr><tr><td> ملقط (FST بواسطة دومون AA/11210-10) </td><td> لاستخدامها خلال تشريح الدماغ من الجمجمة وفصل المخيخ عن بقية الدماغ. هذه ضرورية لأن لديهم معلومات سرية مقربة قليلا أن يساعد على التقليل من الأضرار التي لحقت المخيخ خلال تشريح. </td></tr><tr><td> Nutator (فيشر العلمية) </td><td> تستخدم للحفاظ على النسيج في الحركة خلال فترات حضانة. </td></tr><tr><td> 1.5 مل أنبوب (Sarstedt / المكسرات سقف مايكرو أنبوب) </td><td> كل الخطوات من لهtochemistry بروتوكول تجري في هذه microtubes. أسفل تقريب يضمن أن يبقى في المخيخ الحركة. </td></tr><tr><td> مثقب ملعقة (FST/10370-17) </td><td> تستخدم للحفاظ على wholemounts في microtubes بينما الصب بلطف خارج الحل قضى. </td></tr><tr><td> مجهر لايكا MZ16 FA </td><td> تستخدم لفحص تلطيخ wholemount. </td></tr><tr><td> لايكا DFC3000 FX الكاميرا </td><td> تستخدم لالتقاط الصور wholemount. </td></tr></tbody></table> الجدول رقم 1. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td colspan="9"> تقويم مثال لتجربة نموذجية wholemount </td></tr><tr><td> اليوم 1 </td><td colspan="4"> دنت في الإصلاح، درجة حرارة الغرفة، 8 ساعات </td><td colspan="4"> دنت في التبييض، و 4 درجات مئوية، بين عشية وضحاها </td></tr><tr><td> اليوم 2 </td><td colspan="3"> 100 MeOH٪، درجة حرارة الغرفة، 2X، كل 30 دقيقة </td><td colspan="3"> 100 MeOH٪، تجميد / الذوبان، 4X، 30 دقيقة min/15 </td><td colspa<html> ن = &quot;2&quot;&gt; MeOH 100٪، -80 درجة مئوية، بين عشية وضحاها </td></tr><tr><td> اليوم 3 </td><td> 50٪ MeOH/50٪ PBS، درجة حرارة الغرفة، 60-90 دقيقة </td><td colspan="2"> 15٪ MeOH / PBS 85٪، درجة حرارة الغرفة، 60-90 دقيقة </td><td colspan="2"> 100 PBS٪، درجة حرارة الغرفة، 60-90 دقيقة </td><td> 10μg/mL بروتين كاف في برنامج تلفزيوني، درجة حرارة الغرفة، 2-3 دقيقة </td><td> 100 PBS٪، درجة حرارة الغرفة، 3X، 10 دقيقة كل </td><td> PMT، 4 درجات مئوية، بين عشية وضحاها </td></tr><tr><td> يوم 4-5 </td><td colspan="8"> PMT + 1 ° الضد + 5٪ DMSO، 4 درجات مئوية، 48 ساعة </td></tr><tr><td> 6 أيام </td><td colspan="3"> PMT، 4 درجات مئوية، 2-3X، 2-3 ساعات كل </td><td colspan="5"> PMT + 2 ° الضد + 5٪ DMSO، 4 درجات مئوية، 24 ساعة (أو تبدأ خطوات التضخيم مع مجمع ABC) </td></tr><tr><td> يوم 7 </td><td colspan="2"> PMT، 4 درجات مئوية، 2-3X، 2-3 ساعات كل </td><td colspan="3"> PBT، درجة حرارة الغرفة، 2 ساعات </td><td colspan="3"> في احتضان DAB جديدة في برنامج تلفزيوني حتى تلطيخ الأمثل هو تصور </td></tr></tbody></table>_content &quot;&gt; الجدول رقم 2. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td colspan="2"> وصفات (* = إعداد جديد في كل مرة) </td></tr><tr><td> برنامج تلفزيوني (الفوسفات مخزنة المالحة) </td><td> مخزنة 0.1M فوسفات المالحة في الماء منزوع الأيونات. درجة الحموضة 7.2 (أقراص سيغما، P4417) </td></tr><tr><td> PFA (لامتصاص العرق) </td><td> أدلى والمجمدة تخزين كحل 20٪ ومن ثم تخفيفه إلى 4٪ في برنامج تلفزيوني من أجل حل العمل (فيشر العلمية؛ T353) </td></tr><tr><td> دنت ومثبت 3 * </td><td> 4 أجزاء الميثانول 1 dimethylsulfoxide جزء (DMSO؛ فيشر العلمية؛ D159-4) </td></tr><tr><td> دنت في بليتش 3 * </td><td> 4 أجزاء الميثانول 1 dimethylsulfoxide جزء (DMSO؛ فيشر العلمية؛ D159-4) 1 بيروكسيد الهيدروجين جزء 30٪ </td></tr><tr><td> الأنزيمية الهضم </td><td> 10 ميكروغرام / مل من K بروتين (روش تشخيص، 03115828001) في برنامج تلفزيوني. </td></tr><tr><td> PBST </td><td> برنامج تلفزيوني وعلى متنها: 0.1٪ توين-20 (Fisheالبحث العلمي، BP337، ويمكن أيضا أن تستخدم تريتون في مكان توين-20 في جميع الحالات). </td></tr><tr><td> PMT 25 * </td><td> برنامج تلفزيوني وعلى متنها: 2٪ دهن مسحوق الحليب الخالي من الدسم (قرنفل فضل) 0.1٪ توين-20 (فيشر العلمية؛ BP337) </td></tr><tr><td> PBT 25 * </td><td> برنامج تلفزيوني وعلى متنها: 0.2٪ البقري (سيغما؛ B9001S) مصل 0.1٪ توين-20 (فيشر العلمية؛ BP337) </td></tr><tr><td> DAB * </td><td> حل 1 10 ملغ من diaminobenzidine-3،3 (سيغما الدريخ؛ D5905) في 40 مل من برنامج تلفزيوني. إضافة 10 ميكرولتر من بيروكسيد الهيدروجين بنسبة 30٪ لبدء رد فعل). </td></tr><tr><td> ABC حل معقد </td><td> Vectastain مجموعة (المتجهات المختبرات، وشركة؛ PK-4000) </td></tr></tbody></table> الجدول 3.

wholemount immunohistochemistry for revealing complex brain topography

بنية متكررة ومفهومة جيدا الخلوية من المخيخ جعله نظاما نموذج مثالي لاستكشاف تضاريس الدماغ. الكامنة وراء التهندس الخلوي بصورة موحدة نسبيا هي مجموعة معقدة من المجالات مجاور للسهمي من الجينات والبروتينات. وينعكس هذا التقسيم الجزيئية للمخيخ من قبل المنظمة التشريحية والوظيفية للألياف وارد. لنقدر تماما مدى تعقيد منظمة المخيخ نحن صقلها من قبل نهج تلطيخ wholemount لتحليل إنتاجية عالية من العيوب الزخرفة في المخيخ الماوس. هذا البروتوكول يصف بالتفصيل الكواشف، والأدوات، والخطوات العملية التي هي مفيدة للكشف عن أنماط التعبير بنجاح بروتين في المخيخ فأر بالغ باستخدام wholemount المناعية. سلط الضوء على خطوات تثبت هنا فائدة هذه الطريقة تستخدم للتعبير عن zebrinII / aldolaseC كمثال على الكيفية التي يمكن بها كشف تضاريس الدقيقة للدماغ في تقريرهاأصلي ثلاثي الأبعاد التشكل. كما وصفت هي التعديلات على البروتوكول الذي يسمح لتصور من بروتين تعبير وارد في التوقعات ومخيخات كبير للدراسات المقارنة للالتضاريس الجزيئية. لتوضيح هذه التطبيقات، يتم تضمين البيانات من تلطيخ وارد من المخيخ الفئران.

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Wholemount Immunohistochemistry for Revealing Complex Brain Topography

ويتم تنظيم الطوبوغرافي الدوائر العصبية في المقصورات وظيفي مع التشكيلات الجزيئية محددة. هنا، ونحن نقدم الخطوات العملية والتقنية للكشف عن العالمية تضاريس الدماغ باستخدام تنوعا wholemount نهج تلطيخ المناعى. نحن إثبات فائدة من طريقة استخدام التهندس الخلوي مفهومة جيدا والدوائر المخيخ.

Wholemount المناعية للكشف عن تضاريس دماغية معقدة

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

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15.5K Views.  Albert Einstein College of Medicine, Yeshiva University. ويتم تنظيم الطوبوغرافي الدوائر العصبية في المقصورات وظيفي مع التشكيلات الجزيئية محددة. هنا، ونحن نقدم الخطوات العملية والتقنية للكشف عن العالمية تضاريس الدماغ باستخدام تنوعا wholemount نهج تلطيخ المناعى. نحن إثبات فائدة من طريقة استخدام التهندس الخلوي مفهومة جيدا...

Video: Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

المنطقة Subventricular اون الوجه : يلطخ Wholemount والتدفق البطانة العصبية

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Revealing Neural Circuit Topography in Multi-Color

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

الكشف عن العصبية علم الطوبوغرافيا في حلبة لون موضوع

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

طرق التصوير المناعى والكالسيوم في Wholemount الجرذ الشبكية

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

إصابة الحبل الشوكي الكامل وبروتوكول تشريح الدماغ لاحقا Wholemount<em&gt; في الموقع</em&gt; التهجين في اليرقات البحر الجلكى

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

التمهيدي لالمناعية على نسيج Cryosectioned دماغ الفئران: مثال تلطيخ لالدبقية الصغيرة والخلايا العصبية

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

إعداد أنسجة المخ الرئيسيات غير البشرية لمرحلة ما قبل تضمين المناعية والمجهر الإلكتروني

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

إعداد المقطعين الماهيينيات في شبكية العين للكيمياء المناعية

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

تقييم الحث علي الاكتئاب علي المدى الطويل في شرائح البالغين المخيخار

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

الجمع بين التهجين الفلوري المتعدد في الموقع مع الكيمياء الهيستولوجية المناعية الفلورية على أقسام دماغ الفأر الطازجة المجمدة أو الثابتة

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.