Brains are frozen without prior fixation in 2-methylbutane at about -45°C immediately after removal. The frozen brains then are stored at least overnight and generally not more than two months at -80°C before sectioning. On the day of sectioning, the brains are brought to a cryostat temperature of about -20°C for at least 45 minutes before cutting. We section at a thickness of 20µm beginning with the first section we can take from the rostral pole of the bulbs. We first take a section on a room temperature 22-µm x 22-µm coverglass. The following section is taken and thaw-mounted on a gelatin-subbed microscope slide previously cooled to cryostat temperature. We then discard four sections before repeating the process until we have sectioned the entire olfactory bulb. 

Sectioning tissue is perhaps the most technically demanding step in our research. Critical skills include obtaining the correct plane of section and maintaining section integrity throughout the entire olfactory bulb. The plane of section is perpendicular to the long axis of the olfactory bulb - approximately, but not exactly, in a coronal plane relative to the rest of the brain. (If the brain were first mounted in the cryostat to produce a true coronal section, with bulbs facing outward, one would have to tip the bulbs further downwards to achieve our desired plane.) We judge our plane of section by inspecting sections near the beginning of the anterior olfactory nucleus. If the bulbs are cut in our desired plane of section, the lateral mitral cell layer disappears entirely from one collected slide section to the next, or disappears first at the center of the dorsal-ventral extent of the section. If the lateral mitral cell layer is missing either only the dorsal part or only on the ventral part of the section, then the plane is incorrect. We generally tolerate one or two such intervening sections, but if there are three or more intervening sections, the brain is discarded from the analysis. To be cleared for taking sections, our technicians need to demonstrate the proper plane of section in five consecutive brains, a training period that typically takes weeks for someone with experience sectioning brains. Typically, the plane of section remains consistent once this criterion has been reached. 

On the left is a photograph of a fresh-frozen brain mounted in a cryostat to produce the section angle appropriate for our mapping tools. On the right is a photograph of the same brain at the completion of sectioning.

The sections taken on microscope slides are stained for Nissl substance using cresyl violet dye. The coverglass sections are taped using double-stick tape onto card stock together with a set of 14C-standards (American Radiolabeled Chemicals, Inc., 146A) on a 24-µm x 60-µm coverglass. These sections then are exposed for 10 days to Kodak BioMax MR autoradiography film at room temperature. The films are developed in Kodak GBX developer for 3 minutes, rinsed in water for 30 seconds, and fixed in Kodak GBX fixer for 3 minutes.  

After histology and autoradiography, we have a set of glass microscope slides bearing cresyl violet-stained sections (left) and a developed autoradiography film (right) bearing images of the distribution of radiolabel in sections adjacent to the stained ones, as well as images of standards bearing known amounts of carbon-14.