Simultaneous Visualization of NEAT1_2 and Paraspeckle Proteins by Sequential RNA-FISH and Immunofluorescence

Paraspeckles are membrane-less subnuclear assemblies whose organization depends on the long isoform of nuclear paraspeckle assembly transcript 1 (NEAT1_2), which scaffolds paraspeckle proteins into a core-shell architecture. Disruption of the layered structure within paraspeckles occurs when paraspeckle components are depleted, potentially leading to functional impairment. Here, we describe a protocol for simultaneous visualization of NEAT1_2 middle region and transactive response DNA-binding protein 43 (TDP-43), which are enriched in paraspeckle core and shell, respectively, in U2OS human osteosarcoma cells. Sequential RNA-fluorescence in situ hybridization (RNA-FISH) and immunofluorescence, combined with high-resolution confocal microscopy, enable delineation of the paraspeckle core-shell structure. This method provides a robust approach for probing paraspeckle remodeling across diverse biological conditions.