Bifunctional protein cross-linking reagents improve labeling of cytoskeletal proteins for qualitative and quantitative fluorescence microscopy.

Because permeabilization of the cell membrane is necessary to label intracellular proteins with most fluorescent probes, it is important to optimize the preservation and labeling of the proteins under study. We used qualitative and quantitative fluorescence microscopy to evaluate the effects of six different bifunctional protein cross-linking reagents and several extraction conditions on the labeling of filamentous actin with phalloidin and the immunolabeling of tubulin and gelsolin. The labeling of cytoskeletal and associated proteins can be significantly enhanced by the appropriate combination of bifunctional protein cross-linking reagents and extraction conditions. However, the conditions that give the most intense labeling vary depending on the label used. The greatest intensity of labeling with either phalloidin or antibodies was obtained with the intermediate-length cross-linker DSP. The two-step procedure of cross-linking with DSP and extracting in Triton X-100 in microtubule-stabilizing buffer containing DSP gives maximal labeling with phalloidin. Maximal labeling of gelsolin and tubulin with antibodies is obtained by extracting DSP-cross-linked cells with Triton in Hank's saline containing DSP. Therefore, DSP reproducibly improves preservation of both soluble and filamentous proteins for quantitative and qualitative studies by fluorescence microscopy.