Fluorescence photoconversion of biarsenical-labeled cells for correlated electron microscopy (EM)

Cold Spring Harb Protoc. 2011 Jan 1;2011(1):pdb.prot5548. doi: 10.1101/pdb.prot5548.

Abstract

Correlated light microscopy (LM)/electron microscopy (EM) analysis can be achieved by using biarsenical dyes to fluorescently label tetracysteine-tagged proteins. Once live cell imaging using LM is complete, cellular activity can be halted promptly using a glutaraldehyde-based fixative. Rapid fixation preserves cellular ultrastructure and limits diffusion of reaction products. This protocol provides details on rapid fixation of cells, followed by fluorescence photoconversion of 3,3'-diaminobenzidine tetrahydrochloride (DAB) and sample processing for EM that can be correlated with the live cell LM images.

MeSH terms

  • Arsenicals
  • Cell Line
  • Fluorescent Dyes
  • Image Processing, Computer-Assisted / methods
  • Microscopy, Electron / methods
  • Recombinant Proteins / analysis*
  • Staining and Labeling / methods*

Substances

  • Arsenicals
  • Fluorescent Dyes
  • Recombinant Proteins