By Shinichi Nakagawa, Tetsuro Hirose
This quantity specializes in cytological, biochemical, and molecular organic the right way to establish and view the functionality of every nuclear physique, with an emphasis at the research of lengthy non-coding RNAs. Chapters concentrate on exploring fresh experiences that display how convinced lengthy non protein-coding RNAs collect in particular nuclear our bodies and keep an eye on the functionality of the our bodies through serving as architectural parts or controlling the dynamics of associating protein elements. Written within the hugely profitable tools of Molecular Biology sequence layout, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and key tips about troubleshooting and averting identified pitfalls.
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Extra resources for Nuclear Bodies and Noncoding RNAs Methods and Protocols
46 Luca Giorgetti et al. a far red green overlay b X 0 1024 0 Y 1024 40 d Before correction % of beads 20 0 −100 0 100 −100 0 100 −500 0 500 40 After correction 20 0 −100 0 100 −100 ∆X [nm] 0 ∆Y [nm] 100 −500 0 500 300 250 ∆XYZ [nm] c 200 150 without / with Z post-processing 100 50 0 Before After correction ∆Z [nm] Fig. 1. Far-red and green channels are shown (projections across 6 Z planes) along with their overlay. Scale bar, 1 μm. Images are 1,024 × 1,024 pixel wide. 2). (b) A graphical representation of the transformation that was extracted by the UnwarpJ plugin in ImageJ, which registers the green bead image on the far-red image.
Image acquisition of calibration beads: Choose a region of the coverslip where one monolayer of fluorescent beads was adsorbed, and single beads can be resolved as single subdiffraction objects (Fig. 2a). 5 nm, 100 nm Z spacing (see previous section). After acquiring bead images, start imaging DNA FISH experiments with the same microscope parameters. It is important to acquire beads and experiments in the same imaging session, as multiple factors can influence the amount of chromatic aberrations including mechanical manipulation of turret/objectives, sample temperature, and immersion oil temperature.
Double staining is rather tricky, but the following combinations were shown to work: STAR 440SX (Abberior) + Alexa Fluor 514 and Pacific Orange (Life Technologies) + Alexa Fluor 514. When using another laser line (660 nm) for STED depletion, orange to red dyes are compatible and multicolor staining will be easier. 5. The mounting and anti-fade reagents are essential for successful STED imaging. DAPI and Hoechst should not be included, because they are excited by the strong 592 nm depletion beam.
Nuclear Bodies and Noncoding RNAs Methods and Protocols by Shinichi Nakagawa, Tetsuro Hirose