VIS2FIX: rapid chemical fixation of vitreous sections for immuno-electron microscopy.

Abstract

Immuno-electron microscopy uniquely allows high-resolution localization of proteins in their cellular context. Usually, affinity labeling with an electron-dense marker, e.g., small gold particles, is performed on sections of chemically fixed cells or tissues. In this chapter, we describe two novel protocols, the VIS2FIX methods, for chemical fixation of sections of cryo-immobilized biological samples. This method involves production of thin sections of high-pressure frozen cells that are statically adhered to a TEM grid. Subsequent steps involve chemical fixation of the samples by either the VIS2FIX(H) ("H" for "hydrated") or the VIS2FIX(FS) ("FS" for "freeze substitution") techniques. Following chemical fixation, the samples are ready for immunolabeling. The described methods are fast and efficient, yield excellent preservation of intracellular structures, and offer the possibility to maintain lipids in the sample.