Stimulated emission depletion (STED) super resolution imaging of RNA- and protein-containing domains in fixed cells

• Published in: Methods (San Diego, Calif.) Calif.), 2021-03, Vol.187, p.68-76
• Main Authors: Dumbović, Gabrijela, Sanjuan, Xavier, Perucho, Manuel, Forcales, Sonia-V
• Format: Article
• Language: English
• Subjects: lncRNA; Nuclear architecture; Nuclear domains; STED; Super resolution microscopy
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2020.04.009

•Practical guide to single molecule RNA FISH/IF, STED imaging and image processing.•Reduced post imaging processing steps in comparison to STORM approaches.•High-resolution three-dimensional spatial position determination of subcellular structures. Super resolution microscopy has changed our capability to visualize and understand spatial arrangements of RNA- and protein-containing domains in individual cells. In a previous study, we described a novel lncRNA, Tumor-associated NBL2 transcript (TNBL), which originates from a primate specific macrosatellite repeat. We aimed to describe several aspects of TNBL lncRNA, with one focus being pinpointing its precise location in the nucleus, as well as visualizing its interactions with proteins to deduce its functionality. Using a combination of STimulated Emission Depletion (STED) super resolution microscopy, single molecule RNA (smRNA) FISH against TNBL, and immunofluorescence against SAM68 perinucleolar body, we resolved the spatial complexity of the interaction between TNBL aggregates and SAM68 bodies at the perinucleolar region. Here, we describe protocols for a step-by-step optimized smRNA FISH/IF and STED imaging, detailing parameter settings, and three-dimensional data analysis of spatial positioning of subnuclear structures. These protocols can be employed for single-cell imaging of complex nuclear RNA-protein structures.