Intracellular filling in fixed brain slices using Miniruby, a fluorescent biocytin compound

Biocytin is useful for intracellular filling in living slices because it is soluble, has high electrophoretic mobility and a high affinity for avidin. In fixed slices, however, membrane potential cannot be used to signify that a cell is impaled. Thus, it is necessary to inject a fluorescent molecule so that impalement and filling can be visually monitored. As biocytin does not fluoresce, it cannot be used by itself in fixed slices. Here, we report that a biocytin-dextran (MW 10 kDa and 40 kDa) compound, Miniruby (MR), is a useful intracellular marker for injecting neurons in fixed slices. Fixed slices (200-400 microns) of adult rat olfactory bulb, piriform cortex, midbrain periaqueductal gray and locus coeruleus were used. Slices were stained by 0.001% ethidium bromide so that cell bodies could be visualized. The slices were imaged and filled using a specially designed hinged, epi-fluorescent microscope. A cell was impaled with a pipette containing 1-5% MR; positive pulsed constant current (1-5 nA; 300-400 ms on and 600-700 ms off; approximately 10 min) was applied until the fine dendrites were brightly fluorescent. Slices were post-fixed for 6-12 h, then reacted by a conventional ABC-DAB protocol. Miniruby has several advantages: (1) it is easy to visualize the electrode in relation to the cell bodies; (2) the staining procedure is very sensitive, does not require immunohistochemistry, and the reaction product is light stable; (3) injected neurons, dendrites and initial part of axons are well visualized by bright-field microscopy. It should be possible to analyze MR filled cells at the EM level.