Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California, USA.
Nat Protoc. 2012 Oct;7(10):1808-17. doi: 10.1038/nprot.2012.105. Epub 2012 Sep 13.
Detection of RNAs by in situ hybridization (ISH) is a well-established technique that permits the study of specific RNA expression patterns in tissues; however, not all tissues are equally amenable to staining using the same procedure. Here we describe a protocol that combines whole-mount immunofluorescence (IF) and fluorescence in situ hybridization (FISH) for the simultaneous detection of specific RNA transcripts and proteins, greatly enhancing the spatial resolution of RNA expression in complex, intact fly tissues. To date, we have successfully used this protocol in adult testis, larval male gonads, adult intestine and Malpighian tubules. IF is conducted in RNase-free solutions, prior to the harsh conditions of FISH, in order to preserve protein antigenicity within dissected tissues. Separate protocols are described for mRNA and miRNA detection, which are based on robust digoxigenin (DIG) RNA and locked nucleic acid (LNA) probes, respectively. The combined IF-FISH procedure can be completed in 2 d for miRNA detection and 4 d for mRNA detection. Although optimized for Drosophila, this IF-FISH protocol should be adaptable to a wide variety of organisms, tissues, antibodies and probes, thus providing a reliable and simple means to compare RNA and protein abundance and localization.
原位杂交 (ISH) 检测是一种成熟的技术，可用于研究组织中特定 RNA 表达模式；然而，并非所有组织都可以通过相同的程序进行染色。在这里，我们描述了一种结合全组织免疫荧光 (IF) 和荧光原位杂交 (FISH) 的方案，用于同时检测特定的 RNA 转录本和蛋白质，大大提高了复杂、完整的蝇组织中 RNA 表达的空间分辨率。迄今为止，我们已成功将该方案应用于成年睾丸、幼虫雄性性腺、成年肠道和马氏管。在进行 IF 时，在进行 FISH 之前，要在无 RNA 酶的溶液中进行，以在解剖组织内保留蛋白质抗原性。分别描述了用于检测 mRNA 和 miRNA 的方案，它们分别基于稳健的地高辛 (DIG) RNA 和锁核酸 (LNA) 探针。对于 miRNA 检测，IF-FISH 程序可在 2 天内完成，对于 mRNA 检测，可在 4 天内完成。尽管该 IF-FISH 方案针对果蝇进行了优化，但它应该适用于广泛的生物体、组织、抗体和探针，从而提供了一种可靠且简单的方法来比较 RNA 和蛋白质的丰度和定位。
