Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.