Brain Sectioning Protocol
GUIDELINE
Histology of the brain, for obvious reasons, is a research tool rather than a clinical tool. However, this is still invaluable since the brain tissue from donated brains can provide important insights into the underlying pathology of diseases, such as Alzheimer's, which can help in the development of treatments for future patients.
METHODS
- Working in ice-cold Krebs buffer, dissect the brains and embed them (coronal plane) in 4% low melting point agarose in plastic embedding molds. Place the embedding molds on ice to gel the agarose.
- After the agarose hardens (5 min), remove the embedded brain and agarose and trim for vibratome sectioning. To obtain sections with intact thalamic axons, the brain should be cut at an angle tilted 30° forward from the coronal (i.e., top of plane more anterior than bottom of plane).
- Fill Vibratome well with ice-cold Krebs buffer, and surround well with ice. (Change the Krebs buffer and add more ice between brains.).
- Trim agarose to within 2 mm of brain. Cut thick sections (250-300 µm) and collect sections with spatulas into serum-supplemented medium in a sterile Petri dish on ice.
- Select sections that contain the brain region of interest, and transfer them onto polycarbonate membranes (shinny side up), on the surface of serum-supplemented medium (1 ml) in culture plates. Slices must be in incubator within two hours of initial dissection.
- After incubating slices in serum-supplemented medium for 1-2 hours, exchange for serum-free medium and place back in the incubator.
- Slices can now be used for DiI injection, electroporation, or explant culture.
NOTES
- Slices or explants can be maintained in vitro for 3-4 days, after which contamination and cell death become apparent.
- When possible, all brains should be chilled/cooled in wet ice for at least one-half hour prior to sectioning to enhance the ease and quality of sectioning.
