我们感谢邦妮Phetteplace，RN女士协助在图形FNAB过程的演示视频。

简介系统性淀粉样变性是一种高度可变的疾病，是指外，统称为淀粉样蛋白的各种蛋白质沉积。与β褶配置这些淀粉样纤维的沉积，导致各种临床表现取决于所涉及的器官。系统性淀粉样变性的临床相关的表现是有重要器官如心脏，肝脏，和/或肾的参与时指出。从历史上看，这些涉及机关将活检表现出的淀粉样蛋白的存在。这些温和的侵入性程序进行重大风险，包括出血。脂肪垫抽吸以来向人们展示了一个可靠，无创方法检测系统性淀粉样变性淀粉样蛋白在1。这个过程基本上与检索fibroadipose组织评估缺乏在小血管壁2淀粉样蛋白沉积在局部麻醉下于前腹壁皮下脂肪的吸脂。 淀粉样蛋白在组织鉴定的，最经常使用的方法仍然看到时，刚果红染色切片在偏振光显微镜3下可视化特征的苹果绿色双折射模式。当脂肪垫的愿望是，刚果红渍，可以做到无论是直接涂幻灯片或吸气脂肪组织的细胞块准备。然而，在早期阶段的淀粉样变性的患者有很少的淀粉样蛋白沉积，这大大降低了刚果红染色的细胞块切片4,5灵敏度。通过电子显微镜的脂肪垫抽吸的超微结构评价具有更好的可重复性，并提高了灵敏度 4 。因此，建议向所有为一个单元块的编制和执行电子显微镜 2脂肪垫抽吸。 脂肪垫的愿望是一个相对较低的成本和获得组织诊断系统性淀粉样变性的非侵入性的方法。本文介绍了脂肪垫抽吸过程，来样加工的细节，提交两个刚果红染色和电子显微镜超微结构评价标本。在这段视频中，我们展示了这种重复性好，简单的程序，以获取最佳的诊断材料。 1。执行前脂肪垫活检 A.麻醉，局部地区（图1和2） <ol><li>用酒精棉签或一个特定的机构首选的皮肤清洁剂，清洁皮肤，腹部下腹外侧向中线，脐下（图1）。 </li><li>标记一个约2 × 2英寸的菱形形状的区域，如图1所示，标记笔。 </li><li> 18G针吸约10毫升1％的利多卡因。将一个25G的1.5寸毫针注射器。移除窃听直立的注射器，同时按下活塞，直到液体配药，无气泡存在任何残留空气。 </li><li>麻醉沿线已标记的菱形区域，如图2所示的边界。开始，刚下在A点皮肤插入25G针，小心地推针，皮下点十，拉注射器推杆，以确保您不在范围内的船只。然后慢慢推柱塞，同时撤回针点，而不让皮肤针点（图2A3）一个渗透到利多卡因2.5毫升（约利多卡因¼ 10 mL注射器）。用针在皮肤下的改变对点的Y（图2A4）方向，推动皮下针点Y（图2B1）。一如以往，确认针是不是在船只和免除约2.5毫升利多卡因，一边慢慢退出通过点针一个（图2B4 2B3）。重复类似的步骤，从B点出发，从B点和配药利多卡因皮下X与B到Y（图2C1 2D4）。防止从扎伤出血和乙公司应用无菌衡量一块。 </li><li>您现在可以检查该地区是麻醉轻轻抚摸皮肤内麻醉菱形针尖/棉一块纱布一角，与相邻unanesthetized的皮肤比较。 </li></ol> B.执行脂肪垫抽吸术（图3） （应用局部麻醉前脂肪垫吸性能可能被绕过，根据区域和个人喜好。正确执行前脂肪垫抽吸FNAB程序可以在一个刺完成。然而，根据个别病人的痛阈，来回操纵18G针头在皮下脂肪组织相对distres唱。麻醉方法的描述，这里只有两个25G针头刺破达到的效果，并避免出现改善程序的耐受性疼痛。） <ol><li>到10毫升注射器组装18G 1.5寸毫针。装在注射器中的抓地力适当的应用程序和真空释放（“FNAB握/枪”）的注射器针头大会。 </li><li>针尖插入皮下脂肪内的清洗和麻醉的菱形面积（图3a）。 </li><li>完全缩回的注射器针头插入皮下组织产生真空（图3b）柱塞。 </li><li>保持真空和皮下脂肪（图3C通过3I）游刃于各个方向来回针。每个冲程应尽可能长时间而不让皮肤针来选择针的长度。最大采样是通过改变每一笔划的方向（图3I）。重要的是要保持切向浆膜和皮肤表面平行，以避免腹腔穿刺针的方向。 </li><li> fibroadipose组织聚集在注射器中。一旦足够fibroadipose组织碎片（碎片丰富的血液混合标本高达1毫升）进行检索，完全释放的真空，取出针（图3K）。 </li><li>有病人或助理申请程序方面的纱布垫，以防止血液外渗公司的压力。 </li></ol> 2。标本的处理<ol><li>放置在电子显微镜（图4B）戊二醛溶液至少5-6 fibroadipose组织碎片。 </li><li>根据机构的协议，fibroadipose组织碎片数涂片可能准备通过两个幻灯片之间（图4A）蔓延。 </li><li>剩余材料被允许血块在注射器（这可能需要5-7分钟，凝血时间而定）（图4C）。 </li><li>吸10％的福尔马林从墙上的注射器和自由浮动（图4C2）脱落，使凝结fibroadipose组织材料注射器。取下注射器的柱塞（图4C3）和凝结fibroadipose组织转移到10％福尔马林的容器开口端从注射器喷嘴端（图4C4）对面。 </li><li>适当标记的容器，并提交电​​子显微镜和H＆E节和刚果红染色偏光显微镜下的评价福尔马林戊二醛。如果涂片准备，他们可能会根据个别实验室的协议处理。 </li></ol><img src="/files/ftp_upload/1747/1747fig1.jpg" alt="图1" /> 图1。面积为麻醉前脂肪垫FNAB。 <img src="/files/ftp_upload/1747/1747fig2.jpg" alt="图2" /> 图2。麻醉的局部地区。 <img src="/files/ftp_upload/1747/1747fig3.jpg" alt="图3" /> 图3。执行前脂肪垫FNAB。 <img src="/files/ftp_upload/1747/1747fig4.jpg" alt="图4" /> 图4。加工前脂肪垫吸提交实验室检测淀粉样蛋白沉积。 3。代表性的成果<img src="/files/ftp_upload/1747/1747fig5.jpg" alt="图5" /> 图5。淀粉样蛋白在小血管壁fibroadipose组织碎片。Fibroadipose组织嵌入福尔马林处理，石蜡，用刚果红染色，并通过偏光光学显微镜检查。淀粉样蛋白在小血管壁显示苹果绿双折射（白色箭头）。 <img src="/files/ftp_upload/1747/1747fig6.jpg" alt="图6" /> 图6。缺乏组织淀粉样变性。苹果绿双折射缺席在不同的病人组织无淀粉样变性。蓝双折射的胶原纤维（白色箭头），通常存在于几乎所有的标本。 <img src="/files/ftp_upload/1747/1747fig7.jpg" alt="图7" /> 图7。纤维与淀粉样蛋白在血管壁的是一致的。直，无分支的电子显微镜，随机分散的直径为8-10纳米的纤维，由淀粉样蛋白在血管壁的形成。

<ol><li>Westermark, P., Stenkvist, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+method+for+the+diagnosis+of+systemic+amyloidosis%5BTitle%5D)+AND+%22Arch+Intern+Med%22%5BJournal%5D)">A new method for the diagnosis of systemic amyloidosis.</a> Arch Intern Med. 132, 522-523 (1973).</li>
<li>Devata, S., Hari, P., Markelova, N., Li, R., Shidham, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detecting+AL+Amyloid+in+Abdominal+fat+pad+aspirates%3A+Is+Congo+red+sufficient%3F%5BTitle%5D)+AND+%22Amyloid%22%5BJournal%5D)">Detecting AL Amyloid in Abdominal fat pad aspirates: Is Congo red sufficient?</a> Amyloid. , Forthcoming Forthcoming.</li>
<li>Merlini, G., Bellotti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Mechanisms+of+Amyloidosis%5BTitle%5D)+AND+%22New+Engl+J+Med%22%5BJournal%5D)">Molecular Mechanisms of Amyloidosis.</a> New Engl J Med. 349 (6), 583-596 (2003).</li>
<li>Shidham, V. B., Kumar, N., Cihlar, K., Varsegi, G., Markelova, N., Li, R., Hari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fine+needle+aspiration+of+abdominal+fat+pad+for+diagnosis+of+early+amyloidosis%3A+How+can+the+clinical+role+of+the+test+be+improved%3F%5BTitle%5D)+AND+%22Mod+Pathol%22%5BJournal%5D)">Fine needle aspiration of abdominal fat pad for diagnosis of early amyloidosis: How can the clinical role of the test be improved?</a> Mod Pathol. 20, Suppl ement 2. 1A-380A (2007).</li>
<li>Halloush, R., Lavrovskaya, E., Mody, D., Lager, D., Truong, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+and+typing+of+systemic+amyloidosis%3A+The+role+of+abdominal+fat+pad+fine+needle+aspiration%5BTitle%5D)+AND+%22Cytojournal%22%5BJournal%5D)">Diagnosis and typing of systemic amyloidosis: The role of abdominal fat pad fine needle aspiration.</a> Cytojournal. 6, 24-24 (2010).</li>
<li>Hazenberg, I. I., Rijswijk, C. V. an, H, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnostic+Accuracy+of+Subcutaneous+Abdominal+Fat+Tissue+Aspiration+for+Detecting+Systemic+Amyloidosis+and+Its+Utility+in+Clinical+Practice%5BTitle%5D)+AND+%22Arthritis+Rheum%22%5BJournal%5D)">Diagnostic Accuracy of Subcutaneous Abdominal Fat Tissue Aspiration for Detecting Systemic Amyloidosis and Its Utility in Clinical Practice.</a> Arthritis Rheum. , 54-546 (2006).</li>
<li>Guy, C. D., Jones, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abdominal+fat+pad+aspiration+biopsy+for+tissue+confirmation+of+systemic+amyloidosis%3A+specificity%2C+positive+predictive+value%2C+and+diagnostic+pitfalls%5BTitle%5D)+AND+%22Diagn+Cytopathol%22%5BJournal%5D)">Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls.</a> Diagn Cytopathol. 24, 181-185 (2001).</li>
<li>Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosing+Amyloidosis%5BTitle%5D)+AND+%22Scand+J+Rheumatol+Suppl%22%5BJournal%5D)">Diagnosing Amyloidosis.</a> Scand J Rheumatol Suppl. 24, 327-329 (1995).</li>
<li>Linke, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+Sensitive+Diagnosis+of+Amyloid+and+Various+Amyloid+Syndromes+Using+Congo+Red+Fluorescence%5BTitle%5D)+AND+%22Virchow+Arch%22%5BJournal%5D)">Highly Sensitive Diagnosis of Amyloid and Various Amyloid Syndromes Using Congo Red Fluorescence.</a> Virchow Arch. 436, 439-448 (2000).</li>
<li>Giorgadze, T. A., Shiina, N., Baloch, Z. W., Tomaszewski, J. E., Gupta, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+detection+of+amyloid+in+fat+pad+aspiration%3A+an+evaluation+of+Congo+red+stain+by+fluorescent+microscopy%5BTitle%5D)+AND+%22Diagn+Cytopathol%22%5BJournal%5D)">Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.</a> Diagn Cytopathol. 31, 300-306 (2004).</li>
<li>DeMay, R. M. Fine Needle Aspiration Biopsy. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDeMay%2C+RM+%22The+Art+%26+Science+of+Cytopathology%22">The Art & Science of Cytopathology</a>. , ASCP Press, American Society of Clinical Pathology. Chicago. 465-46 (1996).</li>
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淀粉样变性的诊断通常是实现与受影响的器官，如肾脏，肝脏和/或心脏组织活检。然而，这种方法具有较高的诊断率，是侵入性和可能相关的并发症包括出血2。直肠，牙龈和骨髓活检诊断的首选方法在20世纪60年代6,7,8相对较少侵入。腹部脂肪垫抽吸据报道在1973年作为一种安全，微创，简单，更便宜的组织学诊断为系统性淀粉样变性 1程序。然而，检索到的标本处理的程序和方法的细节是不连贯报道。这视频和文章，描述过程中的步骤一步。 检测淀粉样蛋白在脂肪垫抽吸的方法，同样，也没有得到很好的一些研究比较和评估各种方法 1,5,6,7,8标准化。与刚果红染色前脂肪垫抽吸的评价是与观察者间的重复性较低，尤其是在淀粉样变性早期病例与缺乏淀粉样蛋白沉积5敏感。福尔马林固定石蜡包埋细胞块切片进行免疫组织化学和刚果红色荧光细胞学涂片进行报道，以提高诊断的灵敏度9,10。电子显微镜提高了脂肪垫抽吸4，5 fibroadipose组织小血管壁缺乏淀粉样纤维的检测和鉴定。根据这一信息，本文介绍了标本制备细胞块（刚果红染色细胞块切片在偏光显微镜诊断苹果绿色双折射下的评价，也表明免疫组化作为）和电子显微镜的处理。但是，根据淀粉样标本的评价选择的方法，它可能会受到细胞学涂片制备，细胞块的准备，处理和电子显微镜。有些实验室执行从吸气fibroadipose组织碎片，并准备与刚果红染色研究荧光 10涂片测试。 一般在后期的淀粉样变性，与刚果红偏光显微镜的光学显微镜，可能就足够了，但是，在疾病的刚果红染色与偏光显微镜对细胞块切片早期阶段是那么敏感和可靠的脂肪垫抽吸4,5,11 。 Thioflavin T等其他特殊污渍可使用类似的限制。其他的方法来检测淀粉样蛋白，包括与刚果红偏光显微镜和荧光 10淀粉样蛋白的吸涂片的评价。这种方法在我们的经验是少重现。通过电子显微镜淀粉样蛋白脂肪垫血管的评价克服了这些限制 4,11 。淀粉样蛋白的进一步鉴定已通过免疫电镜 12;然而，这些方法可能不会在非三级护理设置广泛。 用于执行共同愿望程序的针头可划分成细计的大小（21 - 25G），中间（18 - 20G），大（如14G）11 。前脂肪垫的愿望已报告使用变量仪表，从中间（18至20G）不等的罚款 ​​（21到22G）2,10执行。肿块大多是与更精细的表针（如25G）吸气，以取得良好的细胞学涂片检查，以及与更广泛的针头（如18G）来获取额外的样品细胞块的额外的通行证。 前脂肪垫抽吸期间，凝聚力的fibroadipose组织不吸以及更细的针头。细胞块的制备及电子显微镜是重要的评价小血在血管壁上的脂肪抽吸fibroadipose组织碎片。更宽的表针（ ​​如18G）推荐产生诊断提供充足的物质 2,10 。由于程序是可比常规FNAB的，被称为脂肪垫抽吸作为FNAB，尽管更广泛的表针 ，可用于执行5。

A correction was made to <a href="https://www-jove-com.bdigitaluss.remotexs.co/index/details.stp?id=1747">Performing and Processing FNA of Anterior Fat Pad for Amyloid</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Safwan S. Jaradeh
instead of:
Safwan S. Jardeh

Erratum: Performing and Processing FNA of Anterior Fat Pad for Amyloid

酒精棉签，纱布，标志着笔，10毫升注射器X2，1％利多卡因（局部麻醉剂，如果单独使用利多卡因，它会导致灌注后最初的烧灼感。这可以通过使用1:1混合的1％利多卡因和1％的钠双向碳酸盐），18号（G）的1.5英寸针X2（表演FNAB），25G 1 ½英寸针（注射局部麻醉剂），FNA的注射器持有人（“枪”），绷带，小瓶（电子显微镜），戊二醛和活检10％的福尔马林的容器。

performing and processing fna of anterior fat pad for amyloid

从历史上看，心脏，肝脏，肾脏活检，证明在淀粉样变性淀粉样蛋白沉积。由于本病的临床表现是如此的变量和非特异性，这些活组织切片检查相关的风险是太伟大的诊断率。其他网站有一个较低的活检的风险，如皮肤或牙龈，也相对侵入性和昂贵。此外，这些活组织切片检查可能并不总是有足够的淀粉样蛋白沉积，以建立一个诊断。脂肪垫的愿望表现出良好的临床相关性低的成本和最小的发病率。但是，有没有执行此过程或处理吸气标本，从而导致变量和nonreproducible结果的标准化协议。检测组织中淀粉样蛋白的最频繁使用的方​​式是对刚果红染色切片使用偏光显微镜的苹果绿色的双折射。这种技术需要细胞块吸气材料的制备。不幸的是，呈现在淀粉样变性的早期阶段的病人有极少量的淀粉样蛋白，从而大大降低了对刚果红染色细胞块切片的脂肪垫抽吸的敏感性。因此，应利用脂肪垫抽吸，通过电子显微镜超微结构评价，由于其增加了对淀粉样蛋白检测的灵敏度。本文演示了一个简单和可重复性的程序执行前脂肪垫抽吸利用细胞块切片和电子显微镜超微结构鉴定两个刚果红染色淀粉样蛋白的检测。

Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Performing and Processing FNA of Anterior Fat Pad for Amyloid

脂肪垫的愿望是首选，微创，成本低的方法，其他方法相比，检测诊断系统性淀粉样变性淀粉样蛋白。此视频文章演示了脂肪垫抽吸标本为最佳的诊断结果进行适当的处​​理与执行程序的轮廓。

表演和加工为淀粉样蛋白前脂肪垫活检

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

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JoVE Journal

Medicine

55.1K 次观看.  Medical College of Wisconsin. 脂肪垫的愿望是首选，微创，成本低的方法，其他方法相比，检测诊断系统性淀粉样变性淀粉样蛋白。此视频文章演示了脂肪垫抽吸标本为最佳的诊断结果进行适当的处​​理与执行程序的轮廓。

视频: Performing and Processing FNA of Anterior Fat Pad for Amyloid

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

科研

生物化学

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

脂肪库专用SCA1的免疫磁性分离<sup&gt;高</sup&gt;脂肪干细胞（ASCs）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

发育生物学

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

新型原子力显微镜基于生物淘选的形态特殊试剂对TDP-43变型肌萎缩侧索硬化症隔离

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

生物工程

A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. To overcome this hurdle, a rigorous protocol was developed that preserves adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. The protocol also outlines how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in the expression of adipogenic genes. Additionally, it can be adapted to visualize adipocytes by whole-mount immunofluorescence of muscle samples. Finally, this protocol outlines how to perform genetic lineage tracing of Pdgfr&#945;-expressing FAPs to study the adipogenic conversion of FAPs. This protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes, along with confirmation by RT-qPCR, allowing for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, the analysis pipeline described here is the first step to improving our understanding of how FAPs differentiate into intramuscular fat, and provides a framework to validate novel interventions to prevent fat formation.

The replacement of healthy muscle tissue with intramuscular fat is a prominent feature of human diseases and conditions. This protocol outlines how to visualize, image, and quantify intramuscular fat, allowing the rigorous study of the mechanisms underlying intramuscular fat formation.

检查骨骼肌中肌内脂肪形成及其细胞起源的指南

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

脂肪组织中的血管和神经纤维的共染色

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

免疫与感染

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

神经科学

Autologous Microfractured and Purified Adipose Tissue for Arthroscopic Management of Osteochondral Lesions of the Talus

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several studies have focused their attention on mesenchymal stem cells derived from adipose tissue. Adipose-derived stem cells (ADSCs) exhibit morphological characteristics and properties similar to other mesenchymal cells, and are able to differentiate into several cellular lines. Moreover, these cells are also widely available in the subcutaneous tissue, representing 10 - 30% of the normal body weight, with a concentration of 5,000 cells per gram of tissue.
In the presented technique, the first step involves harvesting ADSCs from the abdomen and a process of microfracture and purification; next, the surgical procedure is performed entirely arthroscopically, with less soft tissue dissection, better joint visualization, and a faster recovery compared with standard open procedures. Arthroscopy is characterized by a first phase in which the lesion is identified, isolated, and prepared with microperforations; the second step, performed dry, involves injection of adipose tissue at the level of the lesion.
Between January 2016 and September 2016, four patients underwent arthroscopic treatment of osteochondral lesion of the talus with microfractured and purified adipose tissue. All patients reported clinical improvement six months after surgery with no reported complications. Functional scores at the latest follow-up are encouraging and confirm that the technique provides reliable pain relief and improvements in patients with osteochondral lesion of the talus.

The aim of this study is to report a protocol for the arthroscopic treatment of osteochondral lesions of the talus using microfractured and purified adipose-derived stem cells.

自体 Microfractured 与纯化脂肪组织在关节镜下治疗距骨软骨病变

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several ...

医学

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

<meta charset="utf8"></head><body>通过免疫荧光染色检测阿尔茨海默病小鼠模型中的淀粉样蛋白β积累

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

通过免疫荧光染色检测阿尔茨海默病小鼠模型中的淀粉样蛋白β积累

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

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.

Adipo: 三维脂肪组织成像的组织清除方法

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

表演和加工为淀粉样蛋白前脂肪垫活检

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用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

脂肪库专用SCA1的免疫磁性分离

新型原子力显微镜基于生物淘选的形态特殊试剂对TDP-43变型肌萎缩侧索硬化症隔离

检查骨骼肌中肌内脂肪形成及其细胞起源的指南

脂肪组织中的血管和神经纤维的共染色

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

自体 Microfractured 与纯化脂肪组织在关节镜下治疗距骨软骨病变

通过免疫荧光染色检测阿尔茨海默病小鼠模型中的淀粉样蛋白β积累

通过免疫荧光染色检测阿尔茨海默病小鼠模型中的淀粉样蛋白β积累

Adipo: 三维脂肪组织成像的组织清除方法