Matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is used to perform mass spectrometric analysis directly on biological samples providing visual and anatomical spatial information on molecules within tissues. A current obscuration of MALDI-IMS is that it is largely performed on fresh frozen tissue, whereas clinical tissue samples stored long-term are fixed in formalin, and the fixation process is thought to cause signal suppression for lipid molecules. Studies have shown that fresh frozen tissue sections applied with an ammonium formate (AF) wash prior to matrix application in the MALDI-IMS procedure display an increase in observed signal intensity and sensitivity for lipid molecules detected within the brain while maintaining the spatial distribution of molecules throughout the tissue. In this work, we investigate the viability of formalin-fixed tissue imaging in a clinical setting by comparing MALDI data of fresh frozen and postfixed rat brain samples, along with postfixed human brain samples washed with AF to assess the capabilities of ganglioside analysis in MALDI imaging of formalin-fixed tissue. Results herein demonstrate that MALDI-IMS spectra for gangliosides, including GM1, were significantly enhanced in fresh frozen rat brain, formalin-fixed rat brain, and formalin-fixed human brain samples through the use of an AF wash. Improvements in MALDI-IMS image quality were demonstrated, and the spatial distribution of molecules was retained. Results indicate that this method will allow for the analysis of gangliosides from formalin-fixed clinical samples, which can open additional avenues for neurodegenerative disease research.