Immersion fixation with Carnoy solution for conventional immunohistochemical detection of particular N-methyl-D-aspartate receptor subunits in murine hippocampus.

Abstract

Immunoblotting analysis revealed heterologous distribution profiles of the N-methyl-D-aspartate (NMDA) receptor subunits GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 in membrane preparations of different murine brain structures, with exclusive detection of GluR epsilon-3 subunit in cerebellar preparations. Mice were anesthetized and perfused with 4% paraformaldehyde (PA), Zamboni, or Carnoy solution for subsequent immunohistochemical detection of these NMDA receptor subunits. In coronal brain sections from animals perfused with 4% PA and Zamboni solutions, high immunoreactivity was detected exclusively with GluR zeta-1, GluR epsilon-1, GluR epsilon-2, and GluR epsilon-3 subunits in the pyramidal and dentate granular layers, where the GluR epsilon-3 subunit is supposed to be absent. By contrast, high immunoreactivity was detected with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2, but not GluR epsilon-3, subunits in the strata oriens and radiatum of the CA1 subfield without immunoreactivity along the pyramidal and granular layers when sections were prepared by immersion fixation with Carnoy solution after dissection from brains of mice decapitated. On these sections, relatively high immunoreactivity was found also with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the stratum lacunosum-moleculare of the CA1 region, the strata oriens, radiatum, and lacunosum-moleculare of the CA3 region, and the stratum moleculare of the dentate gyrus, respectively. The systemic administration of kainate led to significant decreases in immunoreactivity to GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the CA1 and CA3 subfields on brain sections prepared by immersion fixation with Carnoy solution. These results suggest that immersion fixation with Carnoy solution may be suitable and appropriate for reproducible and quantitative immunohistochemical detection of particular NMDA receptor subunits in murine hippocampus.