Freeze-substitution for morphological and immunocytochemical studies in insects

Methods of plunge freezing and freeze-substitution (FS) for insect antennae and similar body appendages are described. In these more or less cylindrical specimens, usually a layer below the cuticular surface of 10-15 microns thickness is well preserved without freezing damage, further inwards ice-crystal ghosts of increasing size are encountered, but in the very centre of antennal branches (diameter approximately 80 microns) of the silkmoth, Bombyx mori, freezing damage is usually reduced again. The frost-hardy species, Poecilocampa populi and Boreus hiemalis, exhibit regions free from freezing damage up to 40 microns below the cuticular surface. Secondary freezing damage in silkmoth sensory hairs is observed only after deliberately warming the specimens to -43 degrees C for >> 10 min before FS. Secondary artefacts due to the substitution process are investigated by comparison with freeze-etching and by comparing different FS media and protocols. Methanol is not recommended as a substitution medium for insect specimens. Structures particularly liable to substitution damage are the stimulus-conducting pore tubules of olfactory sensilla and the receptor cell membrane. Extraction of soluble components is more likely with pure organic solvents without added chemical fixing agents and with prolonged substitution at elevated temperatures. Such extraction may also be a possible artefact with soluble antigens in immunocytochemical studies. A review is given of the major achievements attained with these techniques is insect functional morphology and immunocytochemistry.