Calcium signaling protocols (Methods in Molecular Biology by David G. Lambert PDF

By David G. Lambert

David Lambert and a panel of major gurus current quite a lot of experimental protocols for learning Ca2+ signaling. those optimized innovations conceal the extra universal functions, together with 45Ca2+ flux measurements, and simple fluorometric expertise, in addition to extra subtle equipment, together with confocal microscopy and subcellular Ca2+ imaging. There also are tools - mostly according to fluorescence dimension - to figure out Ca2+ channel job and the discharge of Ca2+ from intracellular shops. Calcium Signaling Protocols deals modern researchers conveniently reproducible laboratory equipment that give the chance to envision the calcium signaling technique intimately in a number of cells of animal and plant starting place.

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Extra resources for Calcium signaling protocols (Methods in Molecular Biology Vol.114)

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5. Cobbold, P. H. and Rink, R. J. (1987) Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochem. J. 248, 313–328. 6. Hirota, K. and Lambert, D. G. (1997) A comparative study of L-type voltage sensitive Ca2+ channels in rat brain regions and cultured neuronal cells. Neurosci. Lett. 223, 169–172. 7. Brezden, C. , Hedley, D. , and Rauth, A. M. (1994) Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes. Cytometry 17, 343–348.

The phenomenon known as scatter occurs when randomly deflected light deviates many times before it finally impinges on the detector. When these problems are combined, a loss of sharpness, or blurring, of the image results. The confocal principle (1), however, neatly addresses both these flaws. The principle of confocal optics depends on the presence of two pinhole apertures in the light path. One pinhole aperture produces a sharp, intense point of illumination, the intensity of which falls off either side of a chosen depth within the specimen.

7. 05% Triton X-100 for 10 min at room temperature. 8. Wash in PBS several times until surface tension returns to the surface of the slide. 9. If problems with autofluorescence are suspected or known, the sections may be immersed in poly-L-lysine for 1 h at 4°C to quench the autofluorescence. 10. Thoroughly remove excess liquid from the slides using a vacuum-driven filter pump. 11. Add a sufficient amount of first-stage antibody to each experimental section to ensure that the sections are covered.

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