免疫荧光定义细胞的激光捕获显微切割用于mRNA分析：快速通讯

IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication.

作者信息

Murakami H, Liotta L, Star R A

机构信息

Renal Diagnostics and Therapeutics Unit, and Laboratory of Pathology, National Institutes of Health, Bethesda, Maryland 20892-1268, USA.

出版信息

Kidney Int. 2000 Sep;58(3):1346-53. doi: 10.1046/j.1523-1755.2000.00295.x.

DOI:10.1046/j.1523-1755.2000.00295.x

PMID:10972700

Abstract

BACKGROUND

The next phase of the molecular revolution will bring functional genomics down to the level of individual cells in a tissue. Laser capture microdissection (LCM) coupled with reverse transcription-polymerase chain reaction (RT-PCR) can measure gene expression in normal, cancerous, injured, or fibrotic tissue. Nevertheless, targeting of specific cells may be difficult using routine morphologic stains. Immunohistochemistry can identify cells with specific antigens; however, exposure to aqueous solutions destroys 99% of the mRNA. Consequently, there is an overwhelming need to identify specific tissue cells for LCM without mRNA loss. We report on a rapid immunofluorescent LCM (IF-LCM) procedure that allows targeted analysis of gene expression.

METHODS

A LCM microscope was outfitted for epifluorescence and light level video microscopy. Heat filters were added to shield the image intensifier from the laser. Frozen sections were fluorescently labeled by a rapid one minute incubation with anti-Tamm-Horsfall antibody and an ALEXA-linked secondary antibody. Fluorescently labeled thick ascending limb (TAL) cells were detected by low light level video microscopy, captured by LCM, and mRNA was analyzed by RT-PCR for basic amino acid transporter, Tamm-Horsfall protein, and aquaporin-2.

RESULTS

The immunofluorescently identified TAL could be cleanly microdissected without contamination from surrounding tubules. The recovery of RNA following rapid immunofluorescence staining was similar to that obtained following hematoxylin and eosin staining, as assessed by RT-PCR for malate dehydrogenase.

CONCLUSIONS

We conclude that the new apparatus and method for the immunofluorescent labeling of tissue cells targeted for LCM can isolate pure populations of targeted cells from a sea of surrounding cells with highly acceptable preservation of mRNA. Since the TAL is minimally injured following ischemia, identification of the different responses between TAL and surrounding tissue in damaged kidneys may provide new therapeutic targets or agents for the treatment of acute renal failure.

摘要

背景

分子革命的下一阶段将把功能基因组学带入组织中单个细胞的层面。激光捕获显微切割（LCM）与逆转录聚合酶链反应（RT-PCR）相结合，可用于测量正常、癌性、损伤或纤维化组织中的基因表达。然而，使用常规形态学染色来靶向特定细胞可能具有挑战性。免疫组织化学能够识别带有特定抗原的细胞；然而，暴露于水溶液会破坏99%的mRNA。因此，迫切需要在不损失mRNA的情况下识别用于LCM的特定组织细胞。我们报告了一种快速免疫荧光LCM（IF-LCM）程序，该程序可实现对基因表达的靶向分析。

方法

为LCM显微镜配备落射荧光和光水平视频显微镜。添加热滤光片以保护图像增强器免受激光影响。通过与抗Tamm-Horsfall抗体和ALEXA标记的二抗进行快速一分钟孵育，对冰冻切片进行荧光标记。通过低光水平视频显微镜检测荧光标记的厚升支（TAL）细胞，用LCM捕获，然后通过RT-PCR分析碱性氨基酸转运蛋白、Tamm-Horsfall蛋白和水通道蛋白-2的mRNA。