نشكر J. Sedgewick للمساعدة في وضع المجهر مبائر لالتقاط الصور من الحبوب فضية ، المدعومة جزئيا من خلال المنح البحثية NIH AI48484 (ATH) ، AI20048 (RA) ، وAI066314 (قانون القضاء العسكري) ، المركز الوطني لبحوث الموارد المنحة. RR00169 (CNPRC ، جامعة كاليفورنيا ، ديفيس).

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	<li>Altman, J. D., Moss, P. A. H., Goulder, P. J. R., Barouch, D. H., McHeyzer-Williams, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274, 94-96 (1996).</li><li>Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+Edge:+In+situ+tetramer+staining+of+antigen-specific+T+cells+in+tissues.">Cutting Edge: In situ tetramer staining of antigen-specific T cells in tissues.</a> Journal of Immunology. 165, 617-61 (2000).</li><li>Qingsheng, L. i., Skinner, P. J., Ha, S. -. J., Duan, L., Mattila, T., Hage, A., White, C., Barber, D. L., O'mara, L., Southern, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen-specific+and+infected+cells+in+situ+predicts+outcome+in+early+viral+infection.">Visualizing antigen-specific and infected cells in situ predicts outcome in early viral infection.</a> Science. 323, 1726-1729 (2008).</li><li>Haase, T., Haase, K., Zupancic, M., Sedgewick, G., Faust, R. A., Melroe, H., Cavert, W., Gebhard, K., Staskus, K., Zhang, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perelson+Quantitative+Image+Analysis+of+HIV-1+Infection+in+Lymphoid+Tissue.">Perelson Quantitative Image Analysis of HIV-1 Infection in Lymphoid Tissue.</a> Science. 274, 985-989 (1996).</li><li>Li, Q., Duan, L., Estes, J. D., Ma, Z. M., Rourke, T., Wang, Y., Reilly, C., Carlis, J., Miller, C. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peak+SIV+replication+in+resting+memory+CD4+T+cells+depletes+gut+lamina+propria+CD4+T+cells.">Peak SIV replication in resting memory CD4 T cells depletes gut lamina propria CD4 T cells.</a> Nature. 434, 1148-1152 (2005).</li><li>Li, Q. i. n. g. s. h. e. n. g., Estes, J. a. c. o. b. D., Schlievert, P. a. t. r. i. c. k. M., Duan, L. i. j. i. e., Brosnahan, A. m. a. n. d. a. J., Southern, P. e. t. e. r. J., Reilly, C. a. v. a. n. S., Peterson, M. a. r. n. i. e. L., Schultz-Darken, N. a. n. c. y., Brunner, K. e. v. i. n. G., Nephew, K. a. r. l. a. R., Pambuccian, S. t. e. f. a. n., Lifson, J. e. f. f. r. e. y. D., &amp;amp, J. o. h. n. V. C. a. r. l. i. s., Haase, A. s. h. l. e. y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol+monolaurate+prevents+mucosal+SIV+transmission.">Glycerol monolaurate prevents mucosal SIV transmission.</a> Nature. , (2009).</li></ol>

The authors have nothing to disclose.

منذ يستخدم الفيروس RNA كعلامة للخلايا المصابة بالفيروسات في هذا التقرير ، ينبغي لجميع الكواشف قبل التهجين يكون حرا RNAase. وقد تم إجراء تعديل في الموقع التهجين الموصوفة هنا للحفاظ على إشارة من الفلورسنت في تلطيخ tetramer الموقع. منذ radioautographic إشارة من الخلايا المصابة بالفيروس - - الكشف عنها بواسطة التهجين في الموقع هو على عمق حوالي واحد قطره الخلية ، والأجزاء السميكة المستخدمة في تلوين tetramer الوضع الطبيعي أن تكون في recut 5-8 ميكرون سميكة المقاطع. ووصف هذا الأسلوب في الجمع بين رواية tetramers الموقع والتهجين في الموقع (ISTH) هنا يمكن تصور في وقت واحد ، ورسم الخرائط وتحليل العلاقة المكانية محددة الفيروس خلايا CD8 + T والخلايا المصابة بالفيروسات الهدف. ويمكن تطبيق هذه الطريقة لتقييم الخلايا التائية CD8 + يستند اللقاح والممرض في الفيروسات الأخرى غير المضيفة والتفاعلات أسلوب تحليل الصور الموصوفة هنا يمكن أيضا أن تتكيف مع دراسة العلاقات المكانية من السكان أي اثنين خلية التفاعل في الموقع ، على سبيل المثال كنا في الآونة الأخيرة إجراء دراسة الخرائط في النقطه الوسطى من HIV - 1 الاتصال الجنسي في نموذج الرئيسيات غير البشرية من نقص المناعة القردي عدوى الفيروس من قرود المكاك ريسوس 6

حلول والكواشف : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مخزنة PBS - H الفوسفات المالحة التي تحتوي على الهيبارين (100ug/ml أو 18.7U/ml) لكبح RNASes </li><li style=";text-align:right;direction:rtl"> وسائل الاعلام RPMI زراعة الأنسجة التي تحتوي على الهيبارين (100ug/ml أو 18.7U/ml) لكبح RNASes </li><li style=";text-align:right;direction:rtl"> 4 ٪ انخفاض ذوبان agarose في برنامج تلفزيوني </li><li style=";text-align:right;direction:rtl"> برنامج تلفزيوني مع 2 ٪ مصل الماعز العادي (أو مصل من الأنواع التي قدمت فيها الأضداد fluorophore مترافق). </li><li style=";text-align:right;direction:rtl"> برنامج تلفزيوني مع 2 ٪ مصل الماعز العادية وتريتون 0.3 ٪ X - 100 </li><li style=";text-align:right;direction:rtl"> الصنف الأول MHC tetramers مترافق لFITC </li><li style=";text-align:right;direction:rtl"> مكافحة FITC الأضداد ، مثل الأرنب BioDesign مكافحة FITC </li><li style=";text-align:right;direction:rtl"> مترافق Fluorophore المضادة للمفتش الأرنب الذي تم عبر غاية كثف إلى الأنواع الأخرى مفتش للاستخدام مع وضع العلامات المتعددة ، مثل الماعز المضادة للأرنب ، Cy3 </li><li style=";text-align:right;direction:rtl"> مترافق Fluorophore المضادة للمفتش الفأر الذي تم عبر غاية كثف إلى الأنواع الأخرى مفتش للاستخدام مع وضع العلامات المتعددة </li><li style=";text-align:right;direction:rtl"> مخزنة الطازجة PBS بارافورمالدهيد 4 ٪ </li><li style=";text-align:right;direction:rtl"> 0.01M اليوريا </li><li style=";text-align:right;direction:rtl"> الجلسرين / الجيلاتين تحتوي 4mg/ml البروبيل غاللاتي </li></ul> المعدات الخاصة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Vibratome </li><li style=";text-align:right;direction:rtl"> مشرط </li><li style=";text-align:right;direction:rtl"> شفرات حلاقة </li><li style=";text-align:right;direction:rtl"> مقص جراحي </li><li style=";text-align:right;direction:rtl"> وكتايت فورية سريعة لاصق تعيين (المنتج # 46551) </li><li style=";text-align:right;direction:rtl"> ملقط </li><li style=";text-align:right;direction:rtl"> # 2 فرش الطلاء الهجن الشعر. يمكن تقليم مع شفرة حلاقة لسمك المطلوب </li><li style=";text-align:right;direction:rtl"> 24 شقة كذلك زراعة الأنسجة لوحات مثل قاع فالكون كتالوج # 353226 </li><li style=";text-align:right;direction:rtl"> نسيج الغرف </li><li style=";text-align:right;direction:rtl"> احباط القصدير </li><li style=";text-align:right;direction:rtl"> شريحة من الورق المقوى مجلد كتالوج فيشر على سبيل المثال # 12-587-10 </li><li style=";text-align:right;direction:rtl"> مبائر مجهر </li></ul> الكواشف IST مرجع سريع PBS - H (الفوسفات مخزنة المالحة مع الهيبارين) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 450 مل 1X PBS </li><li style=";text-align:right;direction:rtl"> 50 مل + 1X PBS الهيبارين 10X </li></ul> 1X PBS + الهيبارين 10X <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 500 مل 1X PBS </li><li style=";text-align:right;direction:rtl"> 500 ملغ مسحوق الهيبارين (العثور على المواد الكيميائية الجافة الجرف التخزين) </li></ul> PBS - H / 2 ٪ NGS (الماعز العادي المصل) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 49 مل PBS - H في أنبوب فالكون </li><li style=";text-align:right;direction:rtl"> 1 خ ع مل (وجدت في aliquots 1 مل -20 في الفريزر) </li></ul> PBS - H / 2 ٪ خ ع / تريتون 0.3 ٪ X - 100 <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 49 مل PBS - H / تريتون 0.3 ٪ X - 100 في أنبوب فالكون </li><li style=";text-align:right;direction:rtl"> 1 خ ع مل (وجدت في aliquots 1 مل -20 في الفريزر) </li></ul> PBS - H / 0.3 ٪ تريتون X - 100 <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 500 مل PBS - H </li><li style=";text-align:right;direction:rtl"> 1.5 مل تريتون X - 100 (هذا هو ثخين العثور على رفوف تخزين المواد الكيميائية الجافة) </li></ul> 4 ٪ LMP Agarose <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 2 غرام من مسحوق LMP agarose </li><li style=";text-align:right;direction:rtl"> 50 مل PBS </li><li style=";text-align:right;direction:rtl"> يهز حتى الميكروويف ، مختلطة حتى يذوب المسحوق. </li></ul> GG - NPG (الجيلاتين مع الجلسرين البروبيل galate <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع زجاجة من الجيلاتين الجلسرين 50 درجة في حمام الماء لتذوب </li><li style=";text-align:right;direction:rtl"> إضافة 0،06 غرام من galate بروبيل (موجود على الرف أشارت مارثا) ، يهز جيدا </li><li style=";text-align:right;direction:rtl"> نضع في حمام مائي ، والهز أحيانا حتى المنحل. </li><li style=";text-align:right;direction:rtl"> قسامة في أنابيب مل 1 (أنابيب البني) </li></ul> 4 ٪ بارافورمالدهيد <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 4 مسحوق بارافورمالدهيد ز (العثور على رفوف تخزين المواد الكيميائية الجافة) </li><li style=";text-align:right;direction:rtl"> وضع حوالي 10 مل PBS - H في الاسطوانة ، إضافة مسحوق PF </li><li style=";text-align:right;direction:rtl"> إضافة بعض الماء حتى يصل حجم حوالي 70 مل </li><li style=";text-align:right;direction:rtl"> إضافة واحد من هيدروكسيد الصوديوم بالقطارة. </li><li style=";text-align:right;direction:rtl"> يحرك حتى المنحل (عادة حوالي 20-30 دقيقة) </li><li style=";text-align:right;direction:rtl"> إضافة حمض الهيدروكلوريك حتى تصل درجة الحموضة بين 7-8. </li><li style=";text-align:right;direction:rtl"> ليصل حجم 100 مل من الماء. </li></ul> اليوريا 0.01 M <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 0،3 ز اليوريا (العثور على رفوف تخزين المواد الكيميائية الجافة) </li><li style=";text-align:right;direction:rtl"> 500 مل ماء </li></ul>

a technique to simultaneously visualize virus-specific cd8+ t cells and virus-infected cells in situ

أرقام ومواقع محددة من فيروس CD8 + ويعتقد أن الخلايا التائية النسبي لأرقام ومواقع أهدافهم الخلية المصابة إلى أن تكون حاسمة في تحديد النتائج التي تتراوح من تطهير للإصابة بالأمراض المزمنة المستمرة. نحن تصف هنا وسيلة لتقييم العلاقات المكانية والكمي بين المناعية المستجيب (E) فيروس محددة الموضع CD8 + الخلايا التائية والأهداف المصابة (T) الذي يجمع في تلطيخ الموقع (IST) tetramer للكشف عن الفيروسات محددة CD8 + الخلايا التائية و التهجين (ISH) للكشف عن الحمض النووي الريبي الفيروسي + الخلايا في الأنسجة حيث المعركة بين الدفاعات المناعية والتهاب يحدث. مزيج من تلطيخ وISH IST ، يختصر ISTH ، وتمكن من تصوير ورسم خرائط المواقع من الخلايا المناعية المستجيب والأهداف ، وتحديد من السهل E : نسب تي. ويمكن بعد هذه المعايير بدورها أن تستخدم لتحديد العلاقات بين القرب المكاني ، وتوقيت وحجم الاستجابة المناعية التي توقع النتائج في وقت مبكر العدوى

Watch this Scientific Journal Video about A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ at JoVE.com

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

وهناك تقنية تجمع في تلطيخ tetramer الموقع والتهجين الموضع (ISTH) تمكن التصور ورسم الخرائط وتحليل القرب المكاني للمحددة فيروس CD8 خلايا تي + لأهداف المصابة بالفيروسات على ، وتحديد العلاقات الكمية بين هذه المستجيبات المناعي والأهداف إلى نتائج العدوى.

تقنية لتصور في الوقت نفسه للفيروسات محددة CD8 + الخلايا T والخلايا المصابة بالفيروسات في الموضع</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

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Research

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Biology

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

12.0K Views.  University of Minnesota. وهناك تقنية تجمع في تلطيخ tetramer الموقع والتهجين الموضع (ISTH) تمكن التصور ورسم الخرائط وتحليل القرب المكاني للمحددة فيروس CD8 خلايا تي + لأهداف المصابة بالفيروسات على ، وتحديد العلاقات الكمية بين هذه المستجيبات المناعي والأهداف إلى نتائج العدوى.

Video: A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes

التنظيمية خلايا T : الإمكانات العلاجية لعلاج رفض الأعضاء والنوع الأول من مرض السكري

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

صاروخ موجه من الخلايا المكونة للدم في نخاع العظام

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

في تقطير التحت خلوية الموقع من خلايا الثدييات وغير ملتصقة منتسب -

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

عزل الخلايا البطانية صمامي

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

مولد الرمع الفحص : خلايا منتسب

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

الوسم نسب خلايا اسماك الزرد مع فلوريسئين الليزر ديكستران Uncagable

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

سم كلوي حقن مكروي في الزرد لنموذج قصور كلوي حاد

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

واجهة المستخدم الرسومية لتتبع بمساعدة برنامج تركيز البروتين في النتوءات الخلوية الديناميكية

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

في الموقع تلطيخ إمونوفلورزنت من البلعمة الذاتية في الخلايا الجذعية العضلات

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...