By Attila Németh
This quantity presents an updated compilation of present methodological ways applied for the exploration of nucleolar constitution and function. Chapters hide a variety of protocols that come with imaging of the nucleolus, research of ribosomal RNA transcription and processing, and genomics and proteomics of the nucleolus. Written within the hugely winning Methods in Molecular Biology series structure, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, effectively reproducible laboratory protocols, and pointers on troubleshooting and warding off recognized pitfalls.
Authoritative and practical, The Nucleolus: tools and Protocols provides scientists with a competent sensible guide to facilitate the research of this nuclear compartment on the complex level.
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Additional info for The Nucleolus: Methods and Protocols
8. Incubate in 2× SSC/50 % formamide for at least 2 h. 9. Dilute the probe to a concentration of 2 ng/μL in molecular biology-grade (deionized) formamide (12 μL per slide). Add an equal volume of 2× Hyb. The final probe concentration is 1 ng/μL. 10. Mix well by pipetting and vortexing. Spin 20 s in a tabletop centrifuge to remove air bubbles. Keep the probe at room temperature. 11. Pipette 20 μL of the probe solution into a hybridization chamber. 12. Take a slide out of the 2× SSC/50 % formamide solution.
The same cells imaged by TEM providing the ultrastructural context of the fluorescent proteins. Scale bars: 1 μm separated by less than 200 nm (resolution inherently limited by the wavelength of the light); enhanced resolution is required to precisely relate fluorescent signal to nucleolar subcompartments. Therefore, the challenge is now to develop CLEM method making use of advanced high resolution fluorescent imaging. The group of A. S. Frangakis in Frankfurt (Germany) published CLEM of a tagged nucleolar protein using Direct Stochastic Optical Reconstruction Microscopy (STORM) .
Carefully, remove the grids from the drop and let it dry protected from dust. 6. Counterstain the sections with 12 min incubation in uranyl acetate 5 % in water, shielded from light. Rinse extensively in milli-Q water. Incubate 30 s in lead citrate in NaOH saturated atmosphere (see Note 14). Rinse extensively in milli-Q water. 7. Evaporate 5 nm of carbon over the sections in order to spread the electron charges during TEM process (see Note 15). 8. Sections are analyzed with a Jeol-JEM 1400 electron microscope.
The Nucleolus: Methods and Protocols by Attila Németh