| Advances in Proximity Ligation in situ Hybridization (PLISH)
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Author:
Date:
2020-11-05
[Abstract] Understanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression in intact tissues, which we have now updated. By recording spatial information for every profiled cell, PLISH enables retrospective mapping of distinct cell classes and inference of their in vivo interactions. PLISH has high sensitivity, specificity and signal to noise ratio. It is also rapid, scalable, and does not require expertise in molecular biology so it can be easily adopted by basic and clinical researchers.
[摘要] [摘要]在发育，维持和疾病的背景下了解组织需要确定单个细胞在其天然体内空间范围内的分子谱。我们开发了一种邻近连接原位杂交技术（PLISH），该技术能够定量测量完整组织中单细胞基因的表达，现已更新。通过记录每个分析细胞的空间信息，PLISH可以回顾性绘制不同细胞类别并推断其体内 互动。PLISH具有很高的灵敏度，特异性和信噪比。它也快速，可扩展，并且不需要分子生物学方面的专门知识，因此基础和临床研究人员可以轻松地采用它。
[背景技术]我们最近开发了一种复用原位称为PLISH（邻位连接杂交技术原位杂交）（Nagendran等人，2018）。PLISH与其他现有的空间转录组学技术不同，因为它结合了高性能，快速多路复用，低成本和技术简单性（Wilbrey -Clark等人，2020年）。可以通过自动计算完整的冷冻或石蜡包埋组织中单细胞表达图谱来分析PLISH结果，它与同时进行的免疫染色兼容。
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| A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry
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Author:
Date:
2020-09-20
[Abstract] Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an ...
[摘要] [抽象]像组蛋白翻译后修饰（PTM）一样，染色质修饰对于调节基因表达和细胞表型的许多其他方面至关重要。液相色谱-质谱联用（LC-MS）已成为最适合大规模分析组蛋白和组蛋白PTM的方法。选定的组蛋白PTM具有已知功能，其异常调节与包括癌症在内的多种疾病有关。但是，组蛋白分析很少用于诊断中，部分是由于通量有限且基于LC-MS的分析的重现性不理想。我们描述了一种使用96孔板进行少于一天的高通量样品制备的工作流程。制备后，使用自动直接进样（DI-MS）方法将样品喷雾到无LC的MS中。每次分析都可以通过45个PTM（甲基化，乙酰化和磷酸化（共151个组蛋白标记）和16个未修饰的组蛋白肽进行组蛋白变体的相对定量。由于没有残留或基于LC的批处理效应，该工作流程允许MS运行少于1分钟，并具有更高的重现性和耐用性。最后，我们描述了一种工程化的肽序列，用于精确监控样品制备的效率，可以在DI-MS运行期间检测到该效率。
[背景] 组蛋白是具有球形头部和N末端尾巴的碱性蛋白质，富含精氨酸和赖氨酸残基。一对典型的组蛋白H2A，H2B，H3和H4（称为核心组蛋白）形成一个八聚体，其周围147 ...
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| Analysis of Generalized Fibrosis in Mouse Tissue Sections with Masson’s Trichrome Staining
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Author:
Date:
2020-05-20
[Abstract] Expansion of fibrous connective tissue and abnormal deposition of extracellular matrix (ECM) are at the basis of many fibrotic diseases. Fibrosis can occur in response to both physiological and pathological cues, including wound healing, tissue remodeling/repair and inflammation. Chronic fibrosis can lead to severe tissue damage, organ failure and death. Assessing the extent of organ fibrosis is crucial for accurate diagnosis of this condition. The use of Masson’s trichrome staining of tissue sections from skeletal muscle is a fast method for detection of morphological alterations indicative of a fibrotic phenotype in this organ. This staining method detects the extent of collagen fibers deposition and, because it employs the combination of three dyes, can also distinguish muscle fibers ...
[摘要] [摘要 ] 纤维结缔组织扩张和细胞外基质异常沉积是许多纤维化疾病的基础。纤维化可响应生理和病理学提示而发生，包括伤口愈合，组织重塑/修复和炎症。慢性纤维化可导致严重的组织损伤，器官衰竭甚至死亡。评估器官纤维化的程度对于准确诊断这种情况至关重要。使用Masson的骨骼肌组织切片进行三色染色是检测指示该器官纤维化表型的形态学改变的快速方法。这种染色方法可检测胶原纤维的沉积程度， 因为它使用三种染料的组合，所以还可以同时区分肌肉纤维（红色）和胶原蛋白（蓝色）和细胞核（黑色）。
[背景] 纤维化是由于慢性炎症，组织损伤/重塑（例如，化学或放射疗法后），持续感染，自身免疫性疾病，过敏反应和癌症而在器官中形成过多的纤维结缔组织。在此过程中，包括胶原蛋白在内的过多的细胞外基质（ECM）成分会沉积并积聚。如果进展，纤维化将变成慢性，最终导致器官衰竭甚至死亡（Rockey 等，2015）。塞弗拉尔类型的纤维化疾病在人类，很多都是未知病因进行了描述。通常受影响的器官是肺，肾，肝，心脏和骨骼肌（Hinderer 等人，2019年; Majo 等人，2019年; Mahdy，2019年）。例如，特发性肺纤维化是与肺组织瘢痕形成有关的常见，进行性和致命性疾病，会逐渐失去充氧能力，导致患者无法正常呼吸（Lederer 等人，2018）。
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