From light to electron microscopic analysis of neurons intracellularly injected in fixed slices with “miniruby”, a fluorescent biocyttn compound

Abstract

Intracellular labeling of neurons in fixed slices is a very useful method for studying morphological structures of neurons both at light and electron microscopic levels. Recently, biocytin has been widely used for intracellular labeling in living slices because this molecule is highly soluble, has high electrophoretic mobility and has high affinity for avidin. However, biocytin cannot be used in fixed slices because in fixed slices membrane potential cannot be used to signify that a cell is impaled. Thus, in fixed slices it is necessary to inject cells with a fluorescent compound so that impalement and filling can be visualized under fluorescent microscope. We have developed a fluorescent biocytin compound, “Miniruby” (MR), dextran-tetramethylrhodamine-biocytin. previously, we showed that mis molecule provides excellent intracellular labels in fixed slices at the light microscopic level. Here, we demonstrate MR can also be visualized at the electron microscopic level.Fixed slices (200-400 ¼m) of adult rat olfactory bulb, piriform cortex and periaqeductal gray were used.