Specimen Preparation

Specimen preparation is, without doubt, the most critical factor to obtaining good fluorescent images. Obviously, the design of a preparation protocol for staining will depend upon the types of fluorescent probes being used. Some probes, for example, are cell permeable and readily cross the plasma membrane; these dyes usually require little preparation and may easily be used for staining living cells. Other probes are cell impermeable (i.e. antibodies) and require more effort. They must either be microinjected into living cells or used to stain cells that have been 'fixed' to retain their structural organization and have had their membranes permeabilized. Below, the various steps and considerations for processing are discussed in detail.

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