Microdissection of the supraoptic nucleus and posterior pituitary gland from fresh unfixed tissue

Abstract

The rat supraoptic nucleus (SON) contains magnocellular neurons that project long axons that terminate in the posterior pituitary gland. To perform molecular characterization of these regions, such as transcriptome and methylome profiling, it is necessary to obtain large quantities of high-quality RNA and DNA. Prior methods to isolate molecular material from these small regions required fixing or freezing and laser microdissection of whole tissue, which can compromise recovery and integrity. We have established a straight-forward method of dissecting out the SON and posterior pituitary gland from fresh, unfixed tissue that allows for the isolation of RNA or DNA without compromising nucleic acid integrity. Furthermore, this method can be used as a framework for the microdissection of any region of the brain to isolate any sensitive material. In this manuscript, we describe step-by-step instructions from the macro scale dissection, to brain sectioning, and finally the microdissection of the appropriate tissue. • Transcardial perfusion without fixative prevents the shortcomings of nucleic acid cross-linking. • A fast method and the maintenance of tissue in ice-cold HBSS during dissection and sectioning prevents nucleic acid degradation. • A vibratome is used for the sectioning of fresh brain tissue without freezing or gelatin embedding (i.e. cryostat or microtome).

Keywords

• Supraoptic nucleus
• Posterior pituitary gland
• Microdissection