Antibodies to defined domains of the light and heavy chains of the motor protein kinesin (from squid axon) have been used for immunolabeling of freeze substituted squid axoplasm. It was necessary to develop and apply cryogenic methods to prevent displacement of soluble kinesin during tissue processing. Initial results with this new method first applied with a conventional polyclonal antibody to kinesin suggested that kinesins are widely distributed in the cytoplasm but several~fold concentrated around cytoplasmic vesicles. New experiments using polyclonal antibodies against a defined protein fragment of the functional head of the kinesin heavy chain confirmed the previous kinesin location. An increase in gold particles over the cytoplasmic level was also seen around mitochondria, ER cisternae, and microtubules. Sections incubated with a polyclonal antibody against squid neurofilament, failed, as expected, to show a selective distribution around vesicles. Distributions of different kinesins are being explored by antibodies which distinguish different kinesin light chain isoforms. This work is expected to elucidate where different members of the kinesin family are found in the axon, leading to a better understanding of how kinesins actually function in the nerve cell.
