Basic Neuroanatomical Methods

Abstract

This unit covers some basic procedures that are common to a wide range of neuroanatomical protocols for brain tissue. Procedures are provided for preparation of unfixed fresh brain tissue as well as for perfusion fixation of animals to obtain fixed neural tissue. A variety of methods for sectioning are described, including frozen sectioning using a cryostat or microtome and sectioning with a vibratome. The choice of sectioning method depends on how the brain has been prepared and what histochemical method is to be used. A fluorescent immunohistochemical method to localize endogenous molecules as well as induced markers such as green fluorescent protein and red fluorescent protein is also provided. Additionally, three post-sectioning procedures are described: defatting of slide-mounted sections, fluorescent Nissl staining, and thionin staining of sections. Finally, support protocols are provided, describing a method for maintaining the correct order of cut tissue, whether rostral to caudal or lateral to medial; a procedure for subbing slides with gelatin, which is necessary in some protocols in order for sections to adhere to slides; and preparation of custom 3D-printed 10- or 20-well tissue plates and trays for subsequent immunostaining. Published 2019. U.S. Government. Basic Protocol 1: Preparation of unfixed fresh-frozen brain tissue Basic Protocol 2: Perfusion fixation Basic Protocol 3: Cryostat sectioning of frozen brain tissue Basic Protocol 4: Sliding-microtome sectioning of fixed brain tissue Basic Protocol 5: Vibratome and Compresstome sectioning Support Protocol 1: Tissue collection in a 1-in-10 series Support Protocol 2: Preparation of gelatin-subbed microscope slides Support Protocol 3: Custom 3D-printed 10- and 20-well tissue plates Basic Protocol 6: Post-sectioning procedures I: Fluorescent immunohistochemical localization Basic Protocol 7: Post-sectioning procedures II: Defatting Basic Protocol 8: Post-sectioning procedures III: Nissl staining Basic Protocol 9: Post-sectioning procedures IV: Thionin staining.

Keywords: cryostat; histology; immunohistochemistry; microtome; neuroanatomy; sectioning; vibratome.