While ToF-SIMS is typically used to localize elemental ions in inorganic materials, it is also successfully utilized since several years to get images of a large variety of organic compounds, such as lipids (up to m/z 1500) at the surface of biological tissue sections. This technique can be associated with histology for medical diagnosis in order to correlate structural features with ion images. The possibility to use the same tissue section for both histology and mass spectrometry imaging would be a major advantage in terms of sample preparation and precision on the histological structure localization. In this study, on the one hand, rat brain sections were stained with Hematoxylin-Eosin (HE) after a ToF-SIMS surface analysis, and on the other hand, the lipid mapping with ToF-SIMS was performed after the HE staining procedure. In the first case, we evidenced that the high vacuum conditions applied in ToF-SIMS imaging did not disturb the staining neither the recognition of the brain structures. In the second case, a cholesterol fragment ion, chosen for imaging, was still detected in the brain structure after HE staining. However, it has not been possible to totally overlay the optical image before the staining with the ionic images after the staining, likely because of a distention of the tissue. Copyright © 2012 John Wiley & Sons, Ltd.