IMPROVED PROCEDURES FOR ELECTRON-MICROSCOPIC VISUALIZATION OF THE CYTOSKELETON OF CULTURED-CELLS

We have developed an improved electron microscopic procedure appropria te for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, cri tical point drying, and platinum/carbon coating of cultured cells and the improvements consistof modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyeth ylene glycol into the extraction medium; cell lysis at room temperatur e; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydratio n and drying; and uranyl acetate treatment during dehydration. As a re sult, we have obtained a greatly improved quality of electron microsco pic images together with a high consistency of results. Long and strai ght actin filaments were clearly seen in stress fibers and newly forme d lamellipodia. Their polarity was distinctly revealed by decoration w ith myosin subfragment 1. Depletion of actin from cytoskeletons by gel solin treatment allowed for better visualization of myosin, intermedia te filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreporte d millipede-like appearance. The suggested procedure was compatible wi th immunogold labeling as demonstrated with an antibody to tubulin. Co rrelative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, produc ing a close resemblance between the two kinds of images. (C) 1995 Acad emic Press, Inc.