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首页> 外文期刊>Brain research. Brain research protocols >Selective capture of endothelial and perivascular cells from brain microvessels using laser capture microdissection.
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Selective capture of endothelial and perivascular cells from brain microvessels using laser capture microdissection.

机译：使用激光捕获显微切割技术从脑微血管中选择性捕获内皮细胞和血管周细胞。

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摘要

Laser capture microdissection (LCM) of the major cell types comprising brain microvessels offers a powerful technology to explore the molecular basis of the blood-brain barrier in health and disease. However, the ability to selectively retrieve endothelial or perivascular cells, without cross-contamination from the other, has proven difficult. Additionally, histochemical methods previously described for use with LCM have not allowed for identification of all the different size branches of the microvascular tree. Here, we describe a double immunostaining method, combining bright-field and fluorescence microscopy, and using an extensive dehydration with xylene, to clearly identify and spatially resolve endothelial from perivascular cells within all size microvascular branches in frozen brain sections. LCM of these sections, coupled with RNA analysis by reverse-transcription polymerase chain reaction, revealed that captured endothelial cells show endothelial markers but no detectable markers for astrocytes or smooth muscle cells/pericytes. Conversely, captured astrocytes or smooth muscle cells/pericytes demonstrate their respective markers, but not those of endothelial cells. This approach has applicability to microarray analysis, thereby enabling global gene profiling of the different cell types along the entirety of the brain microvascular tree.
机译：包括脑微血管在内的主要细胞类型的激光捕获显微解剖（LCM）提供了一项强大的技术，可探索健康和疾病中血脑屏障的分子基础。但是，已经证明难以选择性地回收内皮细胞或血管周细胞而又没有彼此交叉污染的能力。另外，先前描述的与LCM一起使用的组织化学方法不允许鉴定微血管树的所有不同大小的分支。在这里，我们描述了一种双重免疫染色方法，将明视野和荧光显微镜相结合，并使用与二甲苯的广泛脱水作用，以清楚地识别并在空间上分辨冷冻脑切片中所有大小的微血管分支中血管周细胞的内皮细胞。这些切片的LCM，加上通过逆转录聚合酶链反应进行RNA分析，显示捕获的内皮细胞显示出内皮标记，但没有可检测到的星形胶质细胞或平滑肌细胞/周细胞标记。相反，捕获的星形胶质细胞或平滑肌细胞/周质显示出它们各自的标记，但不显示内皮细胞的标记。这种方法适用于微阵列分析，从而使大脑微血管树整体上不同细胞类型的全局基因谱分析成为可能。

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