Specimen Preparation

Permeabilization

Following cross-linking fixation, the plasma membrane must next be extracted to allow entry of cell-impermeable fluorescent probes. This may be achieved by treatment with organic solvents (see above), or by treatment with detergents. Of course, if the cells are to be stained with lipophillic stains, to visualize membranous structures, for example, permeabilization is unnecessary. Likewise, when staining cells for membrane components, such as receptors or extracellular polysaccharides, permeabilization may be disadvantageous as this will allow the probe access to the intracellular membrane compartments through which these molecules are shipped to the cell surface.

There are a wide variety of detergents to choose from, and they differ in their efficiency to extract membranes. Experiments in which cells were microinjected with differently sized fluorescent proteins and then fixed and permeabilized demonstrated that, even after aldehyde fixation, intracellular components can still be extracted by detergent permeabilization³. It is, therefore, a good idea to use the mildest detergent possible that will still allow entry of the probe. Antibodies, for example, are fairly large molecules (around 55 kD), and require extensive permeabilization to allow penetration into thick specimens. Insufficient permeability may also result is high background levels if unbound antibody cannot be washed out sufficiently. Smaller probes, such as nucleic acid-specific dyes, do not require extensive permeabilization and readily diffuse into and out of minimally extracted cells.

I have worked with three different detergents in most of my staining protocols. In increasing order of extraction efficiency they are saponin, Triton X-100, and SDS. Saponin is a relatively mild detergent that solvates cholesterol present in the plasma membrane. At low concentrations, internal membranes remain intact. It is useful for labeling smaller molecules that exist in a soluble state within the cytoplasm. It should be prepared as a stock in DMSO, and is typically used at 0.5 to 1 mg/mL. Triton X-100 is probably the most commonly used permeabilization agent for immunofluorescent staining. This detergent efficiently solvates cellular membranes without disturbing protein-protein interactions. Due to its viscosity, it is conveniently prepared as a 20% stock solution in de-ionized water and stored at 4°C. Triton is usually used at concentrations ranging from 0.1 to 1% for permeabilization. SDS is a non-ionic detergent that is commonly used to denature proteins for electrophoresis. It is useful as a permeabilizing agent to induce slight denaturation of fixed cells in order to reveal epitopes which may be masked from an antibody⁴. It may extract small, poorly cross-linked proteins from fixed specimens, and should not be used on samples fixed by precipitation.

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