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一种通过激光捕获微切割提取免疫标记的高质量 RNA 的鼠心肌细胞的方案。

A protocol for extracting immunolabeled murine cardiomyocytes of high-quality RNA by laser capture microdissection.

机构信息

School of Basic and Medical Biosciences, Faculty of Life Sciences & Medicine, Guy's Campus, King's College London, London SE1 1UL, UK.

出版信息

STAR Protoc. 2022 Mar 8;3(1):101231. doi: 10.1016/j.xpro.2022.101231. eCollection 2022 Mar 18.

DOI:10.1016/j.xpro.2022.101231
PMID:35284837
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8914385/
Abstract

We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible.

摘要

我们开发了一种高效、超短的免疫组织化学-激光捕获显微切割（IHC-LMD）方案，该方案允许对多达 250 个单个心肌细胞进行微切割。在 LMD 之前，取出、快速冷冻并冷冻切片小鼠心脏。从 LMD 材料中分离的 RNA 具有高质量的 RNA，可用于基因表达分析和 RNA 测序。该方案的挑战和局限性包括 PEN 载玻片上免疫染色和核 DAPI 染料的可视化，以及尽可能限制 RNA 降解的时间和速度。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/088dfbe76d02/gr13.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/06736e85d0a1/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/b4608fe0c709/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/8b424b4e503d/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/cb2689c79f69/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/064d700c7077/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/ceab30bf95c6/gr7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/315fbc45a9c0/gr8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/ce4ad5ad4af5/gr9.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/665c8693d4ac/gr10.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/80d1bc8f05fd/gr11.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/088dfbe76d02/gr13.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/63ae253463d9/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/f6f75d56a9a2/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/06736e85d0a1/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/b4608fe0c709/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/8b424b4e503d/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/cb2689c79f69/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/064d700c7077/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/ceab30bf95c6/gr7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/315fbc45a9c0/gr8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/ce4ad5ad4af5/gr9.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/665c8693d4ac/gr10.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/80d1bc8f05fd/gr11.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9e66/8914385/088dfbe76d02/gr13.jpg

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