Intracellular localization of proteins is of utmost importance when characterizing protein functions. The most promising approach to perform immunocytochemistry on an ultrastructural level is to do the labeling on biological material which has not been dehydrated or embedded in resin.
For immunogold-labeling we freeze cryoprotected samples, and prepare thin (Tokuyasu-) cryosections from the frozen material. The labeling is then performed on thawed sections. The procedure is very similar to standard immunocytochemistry protocols for fluorescence microscopy, but we usually use protein A coated gold particles (5 nm or 10 nm) to bind to specific primary antibodies instead of flourophores. The dense gold particles are easily recognized as “immunogold” labels in the beam of the transmission electron microscope.
Keeping the sample hydrated during the entire labeling procedure preserves morphology and antigenicity extremely well, thus allowing specific immuno-localization at high resolution with great labeling success rates.
If you want to label your protein of interest please contact Martin Schauflinger. For more information and detailed protocols also see the following literature:
- Webster, P., and Webster, A. (2007). Cryosectioning fixed and cryoprotected biological material for immunocytochemistry. In Electron Microscopy, (Springer), pp. 257–289.
- Slot, J.W., and Geuze, H.J. (2007). Cryosectioning and immunolabeling. Nature Protocols 2, 2480–2491.