Adaptation of a super-sensitive epitope detection technique for the immunoelectron microscopy of titin filaments in vertebrate striated muscle.

ABSTRACT

A super-sensitive epitope-detection technique based on gold-silver intensification was adapted for pre-embedding immunolabelling of titin filaments in vertebrate striated muscle. Indirect immunoelectron microscopy of titin filaments was performed with monoclonal titin antibodies as primary antibodies and Fab anti-mouse IgG conjugated with 1.4 nm gold particles as secondary antibodies. The secondary antibodies penetrated easily into the tissue owing to their reduced size and the very small gold particles. After the labelling procedure, the tissue was fixed in glutaraldehyde. Since the gold particles were not visible by conventional transmission electron microscopy, they were intensified with a silver developing system. Although the particle size varied nonlinearly with the developing time, very fine grain size was achievable. The technique provided super-sensitive detection with excellent contrast and demonstrated epitopes with both strong and weak affinities.