Localization of cholesterol in rat cerebellum with imaging TOF-SIMS Effect of tissue preparation§

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was utilized to address the issue of cholesterol localization in rat cerebellum, a subject not previously investigated. Rat cerebellum was prepared by three different procedures: (1) fixation in formaldehyde, freeze-protection by sucrose, freezing in liquid nitrogen and sectioning by cryoultramicrotomy and drying at room temperature or (2) freezing in liquid nitrogen, cryostat sectioning at 40 8C and drying at room temperature or (3) high-pressure freezing, freeze-fracturing and freeze-drying. The samples were analyzed in an imaging TOF-SIMS instrument equipped with a Bi1–7 +-source. The cholesterol signal (m/z 369 and 385), showed high intensity in the glial cells in white matter and lower intensity in Purkinje cells and in nuclei of granular layer cells. Specimen treated by procedure 1 showed some signs of diffusion of cholesterol in the tissue. Specimen treated by procedure 2 showed freeze-damage of the cells. Specimen treated by procedure 3 showed distinct localization of cholesterol in well preserved tissue. Thus, high-pressure freezing and freezefracturing was used for further characterization of the distribution of cholesterol in rat cerebellum