Abstract text (incl. figure legends and references)
Cell biologist are frequently looking for tools to localize proteins of interest in biological samples. Specific antibodies are used to solve the questions on a fluorescent light microscopy level. But one would prefer to resolve the intracellular structure together with the immunolabeling pattern at higher resolution (e.g. electron microscopy). For IEM, samples are chemically fixed and embedded in a support layer. Ultrathin sections of 70 nm are cut and immunolabelled with the antibody of choice followed by a binding step with an electron dense Gold particle (visible in EM).
The most efficient immunolabelling procedure is the Tokuyasu cryosectioning technique. Hereby a biological sample is fixed with mild chemicals like glutaraldehyde and paraformaldehyde (either directly or by High Pressure Freezing and Automatic Freeze Substitution). After fixation the sample is embedded in a gelatin support, infused in sucrose and frozen in LN2. Thin sections are cut in a ultracryotome at -120˚C using a diamond knife. Sections are retrieved, thawed, placed on EM-grids and immunolabelled followed by EM analysis. Unfortunately, high antigenicity only goes together with mild fixation and therefor good morphology is sacrificed.
The protocols are challenging and skill demanding. The Tokuyasu method can be applied to a large range of samples including small organisms, tissue samples, and cells. Sections can be used for immunofluorescent- and high-resolution Gold labeling. In this presentation the focus will be on the pitfalls and difficulties of Tokuyasu protocols.