Postembedding immunocytochemistry of large sections of brain tissue: an improved flat-embedding technique

Abstract

A method for osmicating, dehydrating, and flat-embedding large slabs of brain tissue in epoxy resin is presented. This permits the production of semithin sections for postembedding immunocytochemistry that are far larger than can be obtained with other embedding approaches. Vibratomed slabs, 50–200 μm thick and as large as 6 × 8 mm are embedded in a ‘soft’ Araldite epoxy. The slabs are laminated onto the flat surface of a pre-cast epoxy slide. After polymerization, the tissue can be studied on the slide as a whole mount to view osmicated fiber tracts, or in experimental tract-tracing studies, to locate retrogradely labeled cells semithin sections are cut. The rigidity of the epoxy slide ensures that the slabs remain flat and are easily removed and mounted for resectioning. Semithin sections are cut using 8 mm wide glass knives or a 6 mm wide diamond knife and are mounted singly or in serial pairs and immunostained using conventional etching and immunoperoxidase techniques. The relative softness of the epoxy permits dozens of semithin sections to be cut from large blocks without appreciably degrading a glass knife edge. After further polymerization the embedment is also compatible with electron microscopy.