Immunoelectron Microscopy

This technique uses antibodies to detect the intracellular location of structures of particular proteins by electron microscopy. Ultra thin sections are labeled with antibodies against the required antigen and then labeled with gold particles. Gold particles of different diameters enable two or more proteins to be studied.

The EM Resource offers post-embedding immunogold labeling of samples in LRW resin. The investigator must supply the primary and secondary antibodies. The investigator should do immunolabeling at the fluorescent light microscopy level before attempting it at the EM level.

The EM Resource also offers cryo equipment for immunolabeling. This consists of the High Pressure Freezing (HPF) apparatus, freeze substitution system, and low temperature cryochamber for low temperature sectioning. The Leica EM PACT2 is a high pressure freezing machine that is designed to freeze samples up to 200-300 um into the specimen without significant ice crystal damage. HPF provides for better antigen retention for immunolabeling.

HPF is followed by freeze substitution or cryo-ultramicrotomy. The Leica EM AFS2 is capable of freeze substitution, progressive lowering of temperature techniques, and low temperature embedding and polymerization of resins for improved preservation of ultra structure and antigenicity. Cryo-ultramicrotomy is performed by the Leica EM FC6 cryochamber. It is designed for low temperature sectioning of samples at temperatures from -15 to -185 degrees C. The Tokuyasu technique for immunolabeling can then be carried out on the cut sections.

Immunogold Procedures

Overview of immunogold labeling procedure
For more detailed class instruction, see training.

The turn-around time for most immunogold experiments will be from 2-4 weeks depending on experiemental parameters.

	Immunoelectron Microscopy