Reliable detection of RNA in hippocampus sections of mice by FISH up to a post-mortem delay of 24 h

IF 2.1 4区生物学 Q4 CELL BIOLOGY Histochemistry and Cell Biology Pub Date : 2024-04-06 DOI:10.1007/s00418-024-02277-x

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Abstract

Proteins can be successfully localized in post-mortem (PM) brain tissue sections if the time until PM tissue sampling is not too long. In this study, we show that this also applies to the localization of RNA and in particular to the RNA of microglia-specific receptor proteins using the probes and the RNAscope^™ Multiplex Fluorescent Detection Kit v2 from Advanced Cell Diagnostics. Brains were removed from killed mice after different PM delays and processed into paraffin sections. In sections of brains from animals whose cadavers had been kept at room temperature (21 °C) before tissue removal, ubiquitously expressed RNAs of genes with low to high expression levels (Polr2a, PPIB, and UBC) were reliably detected in the brain sections even if tissue removal was delayed by up to 48 h. In addition, microglia-specific G protein-coupled receptor RNA (Gpr34, P2ry12) could be reliably assigned to microglia by simultaneous labeling of the microglia with microglia-specific antibodies (Iba1 or P2ry12). Only after a delay of 48 h until tissue removal were the receptor RNA signals significantly lower. The reduction in receptor RNA signals could be delayed if the animal cadavers were stored at 4 °C until the brains were removed. Tissue sections of PM brain samples allow the spatial and cellular localization of specific RNA, at least if the sampling takes place within the first 24 h of PM.

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通过 FISH 对小鼠海马切片中的 RNA 进行可靠检测，检测时间可延长至死后 24 小时

摘要如果距尸检（PM）组织取样的时间不太长，蛋白质就能成功地在尸检（PM）脑组织切片中定位。在本研究中，我们利用先进细胞诊断公司生产的探针和 RNAscope™ Multiplex 荧光检测试剂盒 v2 对 RNA，特别是小胶质细胞特异性受体蛋白的 RNA 进行了定位。经过不同的 PM 延迟后，从被杀死的小鼠体内取出大脑并处理成石蜡切片。在移除组织前尸体保持室温（21 °C）的动物大脑切片中，即使组织移除延迟48小时，也能在大脑切片中可靠地检测到低表达水平到高表达水平基因（Polr2a、PPIB和UBC）的泛表达RNA。此外，通过同时用小胶质细胞特异性抗体（Iba1 或 P2ry12）标记小胶质细胞，可以可靠地将小胶质细胞特异性 G 蛋白偶联受体 RNA（Gpr34、P2ry12）归属于小胶质细胞。只有在延迟 48 小时直至组织移除后，受体 RNA 信号才会明显降低。如果将动物尸体保存在 4 °C，直到取出大脑，受体 RNA 信号的降低可能会延迟。PM 脑样本的组织切片允许对特定 RNA 进行空间和细胞定位，至少如果取样是在 PM 的最初 24 小时内进行的话。

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来源期刊

Histochemistry and Cell Biology 生物-细胞生物学

CiteScore

4.90

自引率

8.70%

发文量

112

审稿时长

1 months

期刊介绍： Histochemistry and Cell Biology is devoted to the field of molecular histology and cell biology, publishing original articles dealing with the localization and identification of molecular components, metabolic activities and cell biological aspects of cells and tissues. Coverage extends to the development, application, and/or evaluation of methods and probes that can be used in the entire area of histochemistry and cell biology.