Ultrarapid freezing — Physical fixation of specimens via ultrarapid freezing includes plunge freezing, slam freezing and spray freezing. Fresh tissues are either frozen on gold blocks with the Leica EM MM80E slam freezer or in liquid propane with a Gentleman Jim plunge freezing device. Cell suspensions are spray-frozen with a homemade spray-freezing device. A new Balzers HPM 010 High Pressure Freezing device has just come online and is capable of freezing samples up to 200 microns without freeze damage occurring. Since these are specialized methods of EM sample preparation, Imaging personnel will perform these procedures.
Freeze substitution — Once samples have been ultrarapidly frozen, they are usually processed for thin sectioning by freeze substitution in a dehydrating agent (acetone or methanol) at —85° C. Freeze substituted samples are then warmed to 4° C, infiltrated in resin and polymerized. It is recommended that Imaging staff carry out this process.
Standard fixation — Routine chemical fixation of tissues and cell cultures includes primary fixation with an aldehyde, secondary fixation with osmium tetroxide, dehydration and infiltration/polymerization in a resin suitable for the project. The entire process takes several days with approximately 8 hours of actual hands-on time.
ltrathin sections — The Imaging staff will cut thin sections (60 — 120 nm) for transmission electron microscopy (TEM) using a Reichert Ultracut E ultramicrotome.
Heavy metal staining — Heavy metal solutions (uranyl acetate, lead solutions and phosphotungstic acid) for positive and negative staining are provided by the facility. Individual users or Imaging personnel can routinely carry out staining procedures for EM.
Immunogold labeling — Localization of antigens on thin sections can be performed by users or by Imaging staff. Gold-labeled secondary antibodies that are available through the facility include goat anti-mouse and goat anti-rabbit antibodies coupled to gold particles of various sizes. Specialized resins required for immunogold localization on thin sections are also provided by the facility.
Carbon coating — A Denton Bench Top Turbo evaporation device is available through the facility for high-vacuum carbon coating EM grids and other samples. This instrument is also available for rotary shadowing, carbon-platinum replicas and glow discharging of EM samples.