Application of “In Vivo Cryotechnique” to Immunohistochemical Detection of Glutamate in Mouse Retina Inner Segment of Photoreceptors

Abstract

In vivo cryotechnique (IVCT) followed by freeze substitution (FS) enabled us to analyze the cells and tissues reflecting living states. In this chapter, we present immunolocalization of glutamate (Glu) in the inner segment (IS) of photoreceptors in the mouse retina with IVCT, which was controversial with conventional fixation methods. Eyeballs from anesthetized mice were directly frozen using IVCT and processed for FS fixation in acetone containing chemical fixatives and embedded in paraffin. Deparaffinized sections were immunostained with an anti-Glu antibody. Strong Glu immunoreactivity was obtained in the specimens prepared by FS with a low concentration of glutaraldehyde, whereas no Glu immunoreactivity was obtained without the chemical fixatives. Thus, it became clear that Glu was immunolocalized in the IS, probably reflecting living states.