Permeabilize Tissue and Cells to Allow Antibody Penetration

Permeabilize Tissue and Cells to Allow Antibody Penetration 

For antibodies to penetrate inside fixed cells, the membranes must be opened with detergents. Membranes are lipid bilayers that have a hydrophilic or water-soluble side facing the cytoplasm and the extracellular space (Fig. 5.4a). The hydrophobic or water-insoluble sides face each other at the center of the membrane. Also, there are transmembrane proteins that interact with the lipids and are held in the membrane. Membranes are barriers because they do not allow water or hydrated molecules to pass. For immunocytochemistry, the membrane must be parhally dissolved to allow antibodies to cross. It is also important that the transmembrane proteins remain cross-linked to other proteins so that they are not washed away (Fig. 5.4b). Detergent will dissolve the membranes but not the transmembrane proteins, which are cross-linked by the fixative to other proteins (e.g., scaffold proteins). For tissue sections, antibodies must penetrate through many cell layers into the center of the section. Achieving this depth of penetration requires removing most of the cell membranes but leaving the proteins so that they can bind antibodies when needed. 

Triton X-100, has methyl groups that form H-bonds and a hydrophobic tail that solubilizes membranes, (b) The ionic detergent, SDS, has a negatively charged group and a hydrophobic tail 

Detergents help dissolve cellular membranes. All detergents are polar lipids that are water soluble with a hydrophilic (polar) ends and can bind the hydrophobic (apolar) ends in the center of the lipid bilayer (Fig. 5.4a). The groups that make up the hydrophilic portion determine the type of detergent. There are two types of detergents, ionic and nonionic (Fig. 5.5). 

Organic solvents like methanol or acetone also can be used to open membranes, but they also act as denaturing fixatives. Solvents are used at -20 C for 10 min, but the use is not recommended. As described in previous sections, denaturing fixatives may cause loss of epitopes in the cells. Other detergent-like agents, such as saponin or digitonin, are used for transient permeabilization of cell cultures, and reversibly insert into and out of plasma membranes next to cholesterol. These agents must be present in all solutions after the fixative and should only be used for electron microscopy immunocytochemistry. 

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