Formalin is a routine fixative facilitating tissue preservation and histopathology. Proteomic techniques require freshly frozen specimens, which are often difficult to procure, and methods facilitating proteomic analysis of archival formalin-fixed brain tissue are lacking. We employed antigen-epitope-retrieval principles to facilitate proteomic analysis of brain tissue that had been fixed and stored in formalin for 3-7 years. Twenty-micrometer-thick cryopreserved OCT-embedded sections from inferior temporal cortex of human (7 years in formalin) or mouse brain specimens (3 years in formalin) were hematoxylin-/eosin-stained. Approximately 16-64-mm2 areas of the tissue sections were manually scraped off slides, or approximately 2 mm2 of human brain cortex was captured off membrane-coated slides using laser microdissection. Tissue was treated using various pH and temperature conditions prior to trypsin digestion and nano-LC-MS/MS. The largest number of proteins were retrieved by solubilization at pH 9 at 95 degrees C for 1 h; treatments at pH 4 or 6 at 25 or 65 degrees C were generally ineffective. Three-year formalin-fixed murine tissue did not yield more proteins compared to human tissue. Use of formalin-fixed tissue for proteomics is an invaluable tool for medical research. The combination of proteomics and microdissection enables selective enrichment and identification of novel, unique, or abundant proteins that may be important in pathogenesis.