By William J. Brown
Targeting new tools and strategies constructed to handle longstanding questions on the Golgi advanced, this quantity explores a various set of chapters, starting from reside and stuck cellphone imaging thoughts to in vitro biochemical reconstitution platforms. every one bankruptcy offers a close set of particular directions, which should still allow somebody to effectively entire the assays. Written for the hugely winning Methods in Molecular Biology sequence software, chapters comprise short introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, easily reproducible laboratory protocols, and suggestions from the specialists on troubleshooting and averting recognized pitfalls.
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Additional info for The Golgi Complex: Methods and Protocols
Because of this, we only use neurons with moderate levels of expression and without signs of somatic Golgi fragmentation. Imaging Golgi Outposts in Fixed and Living Neurons 39 4. To highlight detection of small GOPs (less than 1 μm), either labeled by ectopic expression of fusion proteins or specific antibodies, we routinely used a look-up-table (LUT) called FIRE (scale thermal) available within the LUT table of FIJI ( see also Fig. 2). C. B. is supported by a return home grant from ISN and FONCyT grant (PICT D 2013-1525).
Neurites can be identified by expression of GFP, RFP, CFP, or similar soluble fluorescent proteins, tagged tubulin, or tagged plasma membrane proteins in the transfection mix, and/or endogenous plasma membrane proteins stained with the CF-series dyes (Figs. 3 and 4). 7. Find transfected neurons expressing proteins of interest using the RUSH system (see Note 8). 8. Add 40 μM D-biotin to neurons. Dilute 24 μL of D-biotin (1-mM stock) with neuronal maintenance medium (100 μL) and add to the chamber containing 500 μL of medium.
2. 4 % paraformaldehyde (PFA), 4 % sucrose in PBS buffer. 3. 2 % Triton X-100 in PBS. 4. 5 % bovine serum albumin (BSA) in PBS. 5. 1 % BSA in PBS. 6. FluorSave™ Reagent (or similar medium) for mounting coverslips on microscope slides. 5 Microscopy 1. Conventional confocal microscopes: Zeiss Pascal or Olympus FV300. 2. Spectral confocal microscope: Olympus FV1000. 3. Spinning disk confocal microscope: Olympus IX81 plus Disk Spinning Unit (DSU). , poly-lysine, laminin, and tenascin). Careful preparation of glass coverslips that serve as substrates for neuronal growth is a key step to assure appropriate development of axons and dendritic arbors.