Stimulated emission depletion (STED) super resolution imaging of RNA-and protein-containing domains in fixed cells
Por:
Dumbovic, G, Sanjuan, X, Perucho, M and Forcales, SV
Publicada:
1 mar 2021
Ahead of Print:
1 feb 2021
Resumen:
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.
Filiaciones:
Dumbovic, G:
Univ Colorado, BioFrontiers Inst, Boulder, CO 80309 USA
Sanjuan, X:
Univ Pompeu Fabra, Dept Expt & Hlth Sci, Barcelona, Spain
Ctr Genom Regulat, Adv Light Microscopy Unit, Barcelona, Spain
:
Hlth Sci Res Inst Germans Trias & Pujol IGTP, Program Predict & Personalized Med Canc PMPPC, Canc Genet & Epigenet, Badalona, Spain
Sanford Burnham Prebys SBP Med Discovery Inst, Tumor Initiat & Maintenance Program, La Jolla, CA USA
Forcales, SV:
Univ Barcelona, Fac Med & Hlth Sci, Dept Pathol & Expt Therapeut, Barcelona, Spain
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