This work was supported by a National Institutes of Health grant (GM77311) and a seed grant from The Ohio Cancer Research Associates (to H.A.F.). SM was partially supported by an Up on the Roof fellowship from the Human Cancer Genetics Program of The Ohio State University Comprehensive Cancer Center.

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The authors declare that they have no competing financial interests.

المجهر الكمي في بيولوجيا الخلية ويرتبط عادة مع فحوصات التصوير الخلية الحية، مثل نقل الإسفار الرنين الطاقة (الحنق)، الإسفار الانتعاش بعد photobleaching من (FRAP)، وما إلى ذلك، هناك أمثلة متزايد من علماء الأحياء الخلايا نامية مختلفة المقايسات المجهري الكمية لثابت الخلايا...

تتكون Centrosomes من زوج من centrioles محاطة المواد محيط بالمريكز (PCM). يجري المراكز الرئيسية أنيبيب المنظمة (MTOCs) في خلايا الثدييات، centrosomes بمثابة قطبي مغزل الإنقسامية في تقسيم الخلايا، وبالتالي تساعد على الحفاظ على سلامة الجينوم 1. في الخلايا هادئة (على سبيل المثال، خلال مرحلة G0)، واحد من اثنين من centrioles من الجسيم المركزي، وهي مريكز الأم، وتحويلها إلى هيئة القاعدية لتجميع هدب الابتدائي، عضية الحسية جاحظ الخروج من سطح الخلية 2. مرة واحدة الخلايا إعادة إدخال دورة الخلية، ويتم تفكيكها أهداب الابتدائي وكل مريكز يوجه التجمع من طليعة المريكز في نهايته القريبة أن يستطيل تدريجيا لتشكيل مريكز ناضجة 3. في بداية S-المرحلة، يتم تشكيل بنية تشبه عجلة العربة التي توفر التماثل 9 أضعاف إلى مريكز على سطح كل مريكز القائمة وسوف تصبح قاعدة كل طليعة المريكز. Sas6 رقبعة أمر لا غنى عنه ليتم تجنيده التجمع مريكز لتشكيل محور عجلة العربة 4-6. ثم يتم تجميع البروتينات مريكزي أخرى على عجلة العربة في درجة عالية من التنظيم، الأقرب إلى الطريقة البعيدة 7. بعد الانتهاء بالضبط الازدواجية مريكز، وخلايا تجميع المواد محيط بالمريكز إضافية لبناء اثنين centrosomes وظيفية بحلول نهاية المرحلة G2 8. وبالإضافة إلى المكونات الأساسية مريكزي 9-11، والعديد من البروتينات الأخرى بما في ذلك تحركات، الفسفاتازات، مرافقين، مكونات سقالة، غشاء البروتينات المرتبطة بها والآلات تدهور ترتبط مع centrioles والهيئات القاعدية وPCM في أوقات مختلفة من دورة الخلية 12-16. ويلاحظ في كثير من الأحيان أن مستويات centrosomal من العديد من البروتينات تنظم زمنيا من قبل آليات الاستهداف centrosomal و / أو تدهور proteasomal في centrosomes. الأهم من ذلك أن التقلبات في مستوى centrosomal من عدة بروتينات مثل Plk4، Mps1، Sas6، وCP110 لنقاط ر مختلفة من دورة الخلية ويبدو أن تكون حاسمة لتنظيم التجمع مريكز 5،17-22، وفي حالة Mps1 منع هذا التدهور centrosomal يؤدي إلى تشكيل centrioles الزائدة 19. من ناحية أخرى، فإن الكسور centrosomal من عدة بروتينات أقل عطوب بالمقارنة مع حمامات عصاري خلوي. على سبيل المثال سيرنا بوساطة أسفل تنظيم Centrin 2 (Cetn2) أدى إلى سوى انخفاض معتدل من مستوى البروتين في centrioles على الرغم من انخفاض كبير في مستوياته خلية كاملة 23. ولذا فمن الأهمية بمكان لقياس التغيرات في مستوى البروتينات centrosomal في الجسيم المركزي بدلا من قياس مستويات البروتين خلية كاملة عند تقييم وظائف الجسيم المركزي الخاصة بهم. في هذه الدراسة قمنا بتطوير فحص باستخدام المناعي غير المباشر (معهد التمويل الدولي) لتحديد المستوى النسبي للبروتين في centrosomes. تم تطوير هذا الفحص بشكل خاص لتحليل الخلايا التي هي من عينات مختلفةوبالتالي لا يمكن تصويرها في نفس الوقت. ويمكن لهذه العينات أن تكون الخلايا التي تم التعامل مع الكواشف المختلفة (أي المخدرات مقابل السيطرة)، التي تم جمعها في نقاط زمنية مختلفة (أي نبض مقابل مطاردة)، أو هي في مراحل مختلفة من دورة الخلية. مبدأ هذا الاختبار يكمن في قياس الخلفية تصحيح كثافة الفلورسنت المقابلة لبروتين في منطقة صغيرة وتطبيع تلك القيمة ضد نفسه للبروتين آخر والتي لا تختلف في ظل الظروف التجريبية اختيار المستويات. وقد استخدمت العديد من الدراسات في علم الأحياء الجسيم المركزي مؤخرا مختلف تقنيات المجهر الكمية، في كل الخلايا الحية أو الثابتة، لتحديد وظيفة الجسيم المركزي محددة من البروتينات مرشح 24-27. مماثلة لتلك المقايسات، والتقنية الحالية أيضا يقيس الخلفية تصحيح كثافة مضان من البروتين الاختبار. ومع ذلك، فإن إدراج تطبيع باستخدام معيار الداخلي في هذا الاختبار من المرجح تقديم أكبردقة وثقة في تحليل عينتين المختلفة التي هي على اثنين من coverslips مختلفة. وعلاوة على ذلك، بالإضافة إلى فحص مستوى البروتين في centrosomes، مع تعديلات طفيفة هذه الطريقة يمكن تطبيقها على مجموعة متنوعة من الظروف التجريبية أو في مواقع الخلوية الأخرى. هنا، ونحن لدينا الجمع بين الكمي المجهري الفحص مع استراتيجية نبض مطاردة BrdU لمقارنة الخلايا من مختلف مراحل دورة الخلية. بدلا من استخدام التقنيات القياسية اعتقال دورة الخلية وإطلاق لدراسة مختلف نقاط زمنية دورة الخلية، وخلايا المتزايد بشكل غير متزامن يتم تحضين مع BrdU لتسمية الخلايا في S-المرحلة، ومطاردة الخلايا المسمى لأوقات مختلفة (عادة 4-6 ساعة). ومعظم الخلايا المسمى يكون في S-المرحلة مباشرة بعد النبض. يتم اختيار طول مطاردة وذلك بعد مطاردة، الخلايا المسمى سيكون في أواخر S، G2، أو الانقسام، والتي يمكن التحقق منها من قبل الخصائص المورفولوجية مثل هذا الموقف الفلك من centrosomes فيما يتعلق مجلس التوحيد الوطنىليو، وبعد المسافة بين centrosomes، والتكثيف من الكروموسومات الخ وهكذا، وطول مطاردة يعتمد على متوسط ​​مدة S، M G2 ومرحلة من نوع خلية معينة. منذ هذا النهج يتجنب مثبطات دورة الخلية مثل هيدروكسي يوريا، aphidicolin، نوكودازول، وما إلى ذلك، يتيح تحليل دورة الخلية أكثر أهمية من الناحية الفسيولوجية. وبالتالي، علينا أن نظهر هنا أن الكمية الفحص المجهري مضان وحدها، أو بالاشتراك مع BrdU نبض مطاردة الفحص، هي تقنية بسيطة لكنها قوية لقياس بدقة التغيرات النسبية في مستوى centrosomal من بروتين مرشح خلال دورة الخلية رابط الجأش. قمنا بقياس مستوى centrosomal من VDAC3، وهو البروتين الذي حددنا مؤخرا في centrosomes بالإضافة إلى الميتوكوندريا 16،28، وذلك باستخدام هذه المقايسات. النتائج التي تم الحصول عليها هنا تحقق من ملاحظتنا السابقة أن تجمع centrosomal من VDAC3 ينظمه تدهور، وتختلف أيضا في dependen دورة الخليةر بطريقة 16 والتأكد من صحتها وعلاوة على ذلك إمكانية تطبيق هذا الأسلوب.

<table><tbody><tr><td>Fibronectin</td><td>Sigma</td><td>F8141</td><td>Stock of 1 mg/ml in water</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>MG115</td><td>Sigma</td><td>SCP0005</td><td>Stock of 10 mM in DMSO</td></tr><tr><td>BrdU</td><td>Sigma</td><td>B5002</td><td>Stock of 10 mM in DMSO</td></tr><tr><td>Anti-&amp;#947;-tubulin (mouse monoclonal, clone GTU 88)</td><td>Sigma</td><td>T 6557</td><td>1:200 in IIF blocking buffer&amp;nbsp;</td></tr><tr><td>Anti-VDAC3 (rabbit polyclonal)&amp;nbsp;</td><td>Aviva Systems Biology</td><td>ARP35180-P050</td><td>1:50 in IIF blocking buffer, 1:1,000 in WB blocking buffer</td></tr><tr><td>Anti-Sas6 (mouse monoclonal)&amp;nbsp;</td><td>Santa cruz biotechnology</td><td>sc-81431</td><td>1:100 in IIF blocking buffer</td></tr><tr><td>Anti-Cep135 (rabbit polyclonal)</td><td>Abcam</td><td>ab-75005</td><td>1:500 in IIF blocking buffer</td></tr><tr><td>Anti-BrdU (rat monoclonal)&amp;nbsp;</td><td>Abcam</td><td>ab6326</td><td>1:250 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 350 Goat Anti-Rat IgG (H+L)</td><td>Life technologies</td><td>A21093</td><td>1:200 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody</td><td>Life technologies</td><td>A21202</td><td>1:1,000 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 594 Donkey Anti-Mouse IgG (H+L) Antibody</td><td>Life technologies</td><td>A21203</td><td>1:1,000 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody</td><td>Life technologies</td><td>A21206</td><td>1:1,000 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 594 Donkey Anti-Rabbit IgG (H+L) Antibody</td><td>Life technologies</td><td>A21207</td><td>1:1,000 in IIF blocking buffer</td></tr><tr><td>Anti-&amp;#947;-tubulin (rabbit polyclonal)</td><td>Sigma</td><td>T5192</td><td>1:1,000 in WB blocking buffer</td></tr><tr><td>Anti-&amp;#945;-tubulin (mouse monoclonal, DM1A)</td><td>Sigma</td><td>T9026</td><td>1:20,000 in WB blocking buffer</td></tr><tr><td>Alexa Fluor 680 Donkey Anti-Rabbit IgG (H+L)</td><td>Life technologies</td><td>A10043</td><td>1:10,000 in WB blocking buffer</td></tr><tr><td>Mouse IgG (H&amp;amp;L) Antibody IRDye800CW Conjugated</td><td>Rockland antibodies</td><td>610-731-002</td><td>1:10,000 in WB blocking buffer</td></tr><tr><td>SlowFade Gold Antifade Reagent&amp;nbsp;</td><td>Life technologies</td><td>S36936</td><td>Mounting media</td></tr><tr><td>Round coverslips 12CIR.-1</td><td>Fisherbrand</td><td>12-545-80</td><td></td></tr><tr><td>Olympus IX-81 microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Retiga ExiFAST 1394 IR camera</td><td>QImaging&amp;nbsp;</td><td>32-0082B-238</td><td></td></tr><tr><td>100X Plan Apo oil immersion objective</td><td>Olympus</td><td></td><td>1.4 numerical aperture</td></tr><tr><td>Slidebook software package</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr><tr><td>Odyssey IR Imaging System</td><td>Li-cor Biosciences</td><td></td><td></td></tr><tr><td>Bicinchoninic acid (BCA) assay</td><td>Thermo Scientific</td><td>23227</td><td></td></tr><tr><td>U-MNU2 Narrow UV cube</td><td>Olympus</td><td>U-M622</td><td>Filter</td></tr><tr><td>U-MNU2 Narrow Blue cube</td><td>Olympus</td><td>U-M643</td><td>Filter</td></tr><tr><td>U-MNU2 Narrow Green cube</td><td>Olympus</td><td>U-M663</td><td>Filter</td></tr></tbody></table>

quantitative immunofluorescence assay to measure the variation in protein levels at centrosomes

Centrosomes هي عضيات صغيرة ولكنها مهمة التي تشكل أقطاب الإنقسامية المغزل للحفاظ على سلامة الجينوم أو التجمع أهداب الأساسي لتسهيل مهام الحسية في الخلايا. يجوز تنظيم مستوى بروتين مختلف في centrosomes من في مواقع .cellular أخرى، والاختلاف في مستوى centrosomal من عدة بروتينات في نقاط مختلفة من دورة الخلية ويبدو أن تكون حاسمة لتنظيم السليم التجمع مريكز. قمنا بتطوير مضان الكمي المجهري الفحص الذي يقيس التغيرات النسبية في مستوى البروتين في centrosomes في الخلايا الثابتة من عينات مختلفة، مثل في مراحل مختلفة من دورة الخلية أو بعد العلاج مع مختلف الكواشف. مبدأ هذا الاختبار يكمن في قياس الخلفية تصحيح كثافة الفلورسنت المقابلة لبروتين في منطقة صغيرة، وتطبيع أن قياس ضد نفسه للبروتين آخر لا تختلف تحت ج التجريبية المختارةondition. استخدام هذا الاختبار في تركيبة مع نبض BrdU واستراتيجية مطاردة لدراسة دورات خلية رابط الجأش، لدينا التحقق من صحة كميا الملاحظة الأخيرة في أن ينظم التجمع centrosomal من VDAC3 في centrosomes خلال دورة الخلية، من المحتمل بسبب تدهور بوساطة proteasome وتحديدا في centrosomes.

حددت لدينا دراسات حديثة توطين centrosomal الرواية وظيفة VDAC3، واحدة من porins الميتوكوندريا 16،28. أظهر المناعية من عدة خلايا الثدييات بما في ذلك الخلايا RPE1 باستخدام الأجسام المضادة VDAC3 محددة تلطيخ centrosomal بارز وضعيفة نسبيا تلطيخ الميتوكوندريا. أظهرنا أيضا أن VDAC3 centrosomal يرت...

Watch this Scientific Journal Video about Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes at JoVE.com

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

الكمية المناعي الفحص لقياس التفاوت في مستويات البروتين في Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

quantitative-immunofluorescence-assay-to-measure-variation-protein

Research

JoVE Journal

Biology

14.9K Views.  The Ohio State University. Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard. 

Video: Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

الفلورة الكمية لقياس عالمية مترجمة ترجمة

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemistry

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

تحليل الطيفي تقلب Fluorescence من تفاعلات البروتين البروتين في الخلية جهات الاتصال

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

الآلي الكمي من الإسفار متشابك في<em&gt; C. ايليجانس</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Neuroscience

سير عمل تحليل تلطيخ البروتين النووي في الخلايا الأولية البشرية

A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience.
In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution.
Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different&#160;contexts.

تسليط الضوء على المؤلف: التحليل اللاجيني الشامل للتحقيق في اللدونة الخلوية البشرية والتكيف البيئي باستخدام مقايسات التألق المناعي

This method describes an immunofluorescence protocol and quantification pipeline for evaluating protein distribution with varied nuclear organization patterns in human T lymphocytes. This protocol provides step-by-step guidance, starting from sample preparation and continuing through the execution of semi-automated analysis in Fiji, concluding with data handling by a Google Colab notebook.

خط أنابيب متعدد الاستخدامات لتحليل التغيرات الديناميكية في الأجسام النووية في مجموعة متنوعة من أنواع الخلايا

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the ...

Genetics

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

التحديد الكمي للتوزيع غير متجانسة من البروتين متشابك في الدماغ الماوس استخدام الفلورة

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

Centrosomes التصوير في يطير الخصية

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

سير العمل عالية المحتوى، الكمي خلية فردية من نيون علامات من المجاهر البيانات العالمي، بدعم من برمجيات المصدر المفتوح

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

دليل لمودرن الكمية نيون الغربية التنشيف مع استراتيجيات استكشاف الأخطاء وإصلاحها

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

الكمية المناعي الفحص لقياس التفاوت في مستويات البروتين في Centrosomes

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الفلورة الكمية لقياس عالمية مترجمة ترجمة

تحليل الطيفي تقلب Fluorescence من تفاعلات البروتين البروتين في الخلية جهات الاتصال

الآلي الكمي من الإسفار متشابك في

خط أنابيب متعدد الاستخدامات لتحليل التغيرات الديناميكية في الأجسام النووية في مجموعة متنوعة من أنواع الخلايا

خط أنابيب متعدد الاستخدامات لتحليل التغيرات الديناميكية في الأجسام النووية في مجموعة متنوعة من أنواع الخلايا

خط أنابيب متعدد الاستخدامات لتحليل التغيرات الديناميكية في الأجسام النووية في مجموعة متنوعة من أنواع الخلايا

التحديد الكمي للتوزيع غير متجانسة من البروتين متشابك في الدماغ الماوس استخدام الفلورة

Centrosomes التصوير في يطير الخصية

سير العمل عالية المحتوى، الكمي خلية فردية من نيون علامات من المجاهر البيانات العالمي، بدعم من برمجيات المصدر المفتوح

دليل لمودرن الكمية نيون الغربية التنشيف مع استراتيجيات استكشاف الأخطاء وإصلاحها