Preservation of neuronal ultrastructure in hippocampal slices using rapid microwave-enhanced fixation

Abstract

The goal of this study was to obtain fixation as rapidly as possible and to achieve preservation of neuronal ultrastructure to a depth in hippocampal slices where electrophysiological responses are optimal. This study demonstrates that perfusion quality preservation of in vitro hippocampal slices can be achieved within seconds after removal from the incubation chamber by using microwave (MW)-enhanced immersion in mixed aldehydes. The optimal method was determined to be MW irradiation of the slice for 8-11 s, to a tissue temperature of 35-50 degrees C, during immersion in fixative containing 6% glutaraldehyde and 2% paraformaldehyde. Electron microscopy of these slices revealed ultrastructural preservation that was comparable to hippocampi from animals perfused with mixed aldehyde fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde. Excellent ultrastructural preservation extended to 100-175 microns from the hippocampal slice surface after MW-enhanced fixation and therefore was much deeper than the 8-20 microns that can be obtained by rapid freezing. Hippocampal slices are routinely maintained in vitro for electrophysiological or pharmacological studies. This method of MW-enhanced fixation preserves tissue within seconds after removal from incubation, and should provide good preservation of the hippocampal anatomy that might be associated with in vitro physiology.