The Insect Flight Muscle Sarcomere as a Model System for Immunolocalization

ABSTRACT

Highly ordered insect flight muscle provides an excellent system for coordinated immunolocalization of sarcomeric proteins at increasing levels of resolution, from fluorescent and gold-tagged secondary antibodies to 3- and 5-nm gold directly coupled to Fab fragments. The penetration of antibody probes of various sizes into native and preserved muscle or tissue sections is compared. Factors affecting resolution and labeling efficiency are examined, such as probe size, and removal of uncoupled Fc and gold particles. The quality of preservation and the level of structural detail achieved with ice and plastic embedding media and different labeling methods are explored. We discuss problems encountered at the highest level of immunoelectron microscopy using gold/Fab to visualize the probe in relation to well-contrasted, in situ myosin and troponin molecules in 25-nm-thin epoxy sections by transmission EM.