Elsevier

Methods in Enzymology

Volume 298, 1998, Pages 570-592
Methods in Enzymology

[43] Correlative light and electron microscopy of the cytoskeleton of cultured cells

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      Although the latter methods are powerful in certain aspects, they have their own sources of artifacts and cannot yet replace such rich source of information as properly fixed cells. In application to PREM, we have thoroughly evaluated various fixation protocols using different tests including CLEM to monitor potential artifacts (Korobova and Svitkina, 2010; Svitkina and Borisy, 1998; Svitkina and Borisy, 1999; Svitkina et al., 2003; Svitkina et al., 1995; Yang et al., 2007). These data show that our protocol faithfully preserves the spatial distribution of various cytoskeletal proteins observed by LM in living cells.

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      This method can be used with many types of experimental challenges, making it valuable for investigating the proteins that regulate septin filament organization and remodeling. We used correlative light and electron microscopy (Svitkina, 2016; Svitkina & Borisy, 1998; Svitkina & Borisy, 2006) to confirm that isolated structures were septins. This involves preparing unroofed cortices on coverslips marked with a gold pattern using a finder grid as mask.

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